Multi trigger responsive, surface active lipid nanovesicle aerosols for improved efficacy of paclitaxel in lung cancer

Abstract

The present study focuses on the development of multi-trigger responsive surface active lipid nanovesicles encapsulating paclitaxel with the hypothesis that pulmonary surfactant mimetic lipid vesicles sensitive to temperature and enzyme simultaneously will offer synergistic advantage towards improved therapeutic efficacy of paclitaxel via aerosol administration. The nanovesicles showed a unimodal size distribution of the particles (100–150 nm) and high encapsulation efficiency of paclitaxel (82%). Triggered release of paclitaxel was observed at ∼42 °C in the presence of secretory phospholipase A₂ enzyme with maximum release observed with both the triggers used simultaneously. Since these nanovesicles are intended for aerosol administration in the treatment of lung cancer, they were engineered to have high surface activity and airway patency, in order to mimic pulmonary surfactant functions. High deposition of nanovesicles in the lower impingement chamber of a twin impinger upon nebulization suggested them to be capable of reaching the terminal regions of the lungs. Nanovesicles showed facilitated and ATP dependent active uptake by A549 cells. The cytotoxic potential of the nanovesicles was significantly increased upon simultaneous use of both the triggers with an IC₅₀ of 49.3 nM. Overall, these studies suggest the therapeutic potential and advantages of multi trigger responsive lipid nanovesicles with encapsulated paclitaxel over that of the commercially available form of paclitaxel namely Taxol, and suggests the feasibility of aerosol administration in the treatment of lung cancer and pulmonary metastasis.

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Insight, innovation, integration

The advent of nanotechnology in anticancer drug delivery has substantially lowered the toxicity profile of chemotherapeutics but has offered minimal benefits in terms of antitumor efficacy. We developed temperature and enzyme responsive, surface active lipid nanovesicles for aerosol delivery of paclitaxel towards the treatment of lung cancer with the hypothesis that the use of multiple triggers will be advantageous as compared to a single trigger and therefore will offer synergic beneficial aspects of patient compliant delivery systems, reduced systemic toxicity and improved therapeutic efficacy. These multitrigger responsive and surface active lipid nanovesicles with improved anticancer efficacy, cellular uptake and pulmonary surfactant mimetic properties open new insights towards the improved efficacy and delivery of anticancer drugs as an aerosol in lung cancer.

Introduction

With a 90% mortality rate, lung cancer still remains a major killer among various cancers.¹ This is because of the susceptibility of lungs to both primary as well as secondary metastasized tumors. Traditional treatment strategies for lung cancer include surgical resection of the tumor, radiation therapy and chemotherapy, especially for the advanced cases. All of them pose certain limitations in terms of complete eradication of the tumor. As far as chemotherapy is considered, the main limitation is the non-specificity of the chemotherapeutics, because of which a large proportion of the drug concentrates in normal, healthy tissues resulting in poor biodistribution, decreased anticancer efficacy and increased systemic toxicity. Owing to these issues, a better drug delivery system is required which can be efficiently and specifically targeted to the lungs resulting in improved efficacy and reduced toxicity of the drug. Since the drugs are intended to act topically in the case of lung cancer, their direct administration as aerosol can offer the advantages of increased local concentration and decreased side effects as compared to the systemic administration. Lipid nanovesicles or liposomes have been extensively evaluated as drug carriers and have been proven to be efficient for the delivery of several antineoplastic agents.^2–5 With regard to aerosol delivery of drugs, another advantage with lipid nanovesicles is their easy nebulisation, which can be tuned on the basis of parameters such as lipids used, their concentration, particle size of the nanovesicle and operating conditions of the nebulizer.⁶ Moreover, surface activity of lipid nanovesicles can also be tuned to ensure high airway patency, similar to that of naturally occurring endogenous lung surfactants thereby allowing increased and homogeneous accumulation of drugs even in the terminal airways.⁷ Further, lipid nanovesicle aerosol based drug delivery systems can also offer the advantages of increased compatibility with lung epithelial cells and decreased mucociliary clearance resulting in prolonged residence time.⁵

Lipid nanovesicles have been extensively evaluated and have been found to substantially lower the toxicity profile of anticancer drugs but are often limited in their antitumor efficacy.⁸ Essentially the nanovesicle need to be engineered to exhibit a sustained release profile under normal physiological conditions and a triggered release at the tumor site allowing a preferential accumulation of the drugs at the cancerous sites.⁸ In this regard, researchers have suggested and explored different trigger mechanisms such as pH,⁹ temperature,¹⁰ enzyme,¹¹ ultrasound,¹² light¹³etc. to elicit the site specific triggered release of the drug when required. There has been extensive work indicating the improved therapeutic efficacy with trigger responsive lipid nanovesicles.^14–16 However, to the best of our knowledge there are no reports wherein the combination of these trigger mechanisms has been evaluated in relation to the lipid nanovesicles intended for aerosol administration in lung cancer to understand if such multi trigger responsive lipid nanovesicles gain an edge over single trigger responsive lipid nanovesicles in terms of therapeutic efficacy. Therefore, the objective of the present paper was to design multi-trigger responsive surface active nanovesicles for aerosol therapy and evaluate their advantages over single trigger responsive nanovesicles.

Herein, we developed lipid nanovesicles sensitive to both temperature and secretory phospholipase A₂ (sPLA2), an enzyme overexpressed in the tumor tissue. Temperature sensitivity was imparted by incorporating 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), a thermosensitive lipid with a gel-to-liquid phase transition temperature (T_m) of 56 °C as the primary lipid constituent.¹⁷ Further tuning of the transition temperature was done by incorporating 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), a monounsaturated phospholipid along with an optimized amount of cholesterol in the bilayer resulting in the final transition temperature around 42 °C. Such thermosensitive nanovesicles can elicit the triggered release of drug in response to the heat generated in the processes such as hyperthermia and photothermal therapies. The strategy of enzyme triggered release involves the release of the encapsulated drug as a result of hydrolysis and destabilization of lipid bilayers by sPLA2 enzyme which is normally overexpressed in tumor tissues.¹¹ The sPLA2 enzyme hydrolyzes the ester linkage at sn-2 position of glycerophospholipids thereby producing free fatty acids and lyso-phospholipids which act as the local permeability enhancers both for the tumor cell’s plasma membrane as well as the bilayer of lipid nanovesicles.¹¹ Thus it allows the drug to get released easily and then guides it to the cell’s interior. Paclitaxel, a potent anticancer drug used for the first line treatment of lung cancer was encapsulated into the nanovesicles. It is a diterpenoid pseudoalkaloid and has a unique mechanism of action which involves the stabilization of microtubules resulting in a mitotic arrest in the G2M phase of cell cycle.¹⁸ Its current dosage form, Taxol®, comprises of paclitaxel associated with 50 : 50 (v/v) Cremophor® EL (polyoxyethylated castor oil) and dehydrated alcohol, to increase the solubility. However, this Cremophor® EL based paclitaxel formulation is marked by serious complications causing severe anaphylactoid hypersensitivity reactions, neurotoxicity, cardiotoxicity, nephrotoxicity, hyperlipidaemia, abnormal lipoprotein patterns, erythrocyte aggregation, and peripheral neuropathy.¹⁹ Owing to its highly hydrophobic nature, paclitaxel can be easily encapsulated into the lipid nanovesicles thereby circumventing the Cremophor® associated risks.

In order to test our hypothesis we developed 100–150 nm sized temperature and enzyme (sPLA2) responsive, surface active lipid nanovesicles encapsulating paclitaxel which were characterized for various physiochemical properties. The surface activity of these nanovesicles was tuned to result in high airway patency similar to the naturally occurring pulmonary surfactants. The combination of two trigger mechanisms was found to be synergistically advantageous as compared to a single trigger mechanism as it resulted in a significant increase in the release of paclitaxel under combined trigger conditions which subsequently lead to increased therapeutic efficacy of paclitaxel.

Materials and methods

Materials

1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) with purity >99% were purchased from Avati Polar Lipids, Inc. (Alabaster, USA). Cholesterol (Chol) with purity >99% was purchased from Loba Chemie (Mumbai, India). Paclitaxel (purity >99%) was purchased from Fresenius Kabi India Pvt. Ltd (India). Taxol®, the marketed formulation of paclitaxel, was purchased from Cipla Ltd (India). Dialysis membrane (molecular wt. cutoff 5000–10 000), agarose and DNAse free RNAse were purchased from Himedia Laboratories Pvt. Ltd., Mumbai (India). Dulbecco’s Modified Eagles Medium (DMEM), fetal bovine serum (FBS), antibiotic antimycotic solution, sodium azide, phosphate buffered saline (PBS) and trypsin-EDTA solution were purchased from Himedia Laboratories Pvt. Ltd., Mumbai (India). Sulforhodamine-B, secretory phospholipase A₂ (sPLA2) and proteinase-K were purchased from Sigma Aldrich, Mumbai (India). Rhodamine-6G was purchased from Anaspec Inc. (San. Jose, CA, USA) and a BCA protein assay kit was purchased from Thermo Scientific, Pierce (Rockford, Il, USA). High pressure liquid chromatography (HPLC) grade methanol and chloroform were purchased from Merck, Mumbai (India). All the tissue culture plates and tissue culture flasks were purchased from NUNC (USA). High purity water purified by a Milli Q Plus water purifier system (Milli pore, USA), with a resistivity of 18.2 MΩ cm, was used in all experiments.

Preparation of lipid nanovesicles

Paclitaxel loaded lipid nanovesicles (LN-PTX) were prepared by a modified thin film hydration method,²⁰ with DSPC : POPE in a 4 : 1 molar ratio, phospholipid : Chol in a 7 : 3 molar ratio and paclitaxel : lipid in a 1 : 2 molar ratio. The thin film was hydrated using PBS pH 7.4 with a rotation speed of 100 rpm. The suspension was subsequently sonicated at 20 KHz, 50% amplitude for 2 minutes to form small unilamellar vesicles. Nanovesicles were further extruded through 0.4 μm and 0.2 μm polycarbonate membranes (Avanti Mini Extruder) to obtain a homogeneous size distribution and separate free paclitaxel.¹⁸ The suspension was again centrifuged at 25 000g, 4 °C for 10 minutes and the pellet was reconstituted in PBS pH 7.4 to achieve a final concentration of phospholipids as 1 mg ml⁻¹. Blank nanovesicles (LN-B) were prepared by a similar method without the addition of paclitaxel.

LN-PTX was characterized for size distribution by dynamic light scattering (DLS) using a laser particle analyzer (BI 200SM, Brookhaven Instruments Corporation, USA). The nanovesicles were also characterized for surface charge by determining their zeta potential using a zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corporation, USA). Transmission electron microscopy of the nanovesicles was done as per the negative staining protocol²¹ and images were analyzed by a transmission electron microscope, model: CM200 (Philips) operating at 120 kV. The encapsulation efficiency of paclitaxel in the nanovesicles was determined by breaking them open using methanol and quantifying the drug using reverse phase HPLC (Agilent 1100 Binary LC pump liquid chromatograph).²²

In vitro release of paclitaxel from nanovesicles

An in vitro release profile of paclitaxel from LN-PTX was studied by dialysis bag method²³ under normal physiological conditions (pH 7.4 and 37 °C) and in the presence of single or multiple triggers. Temperature triggered release was studied at 42 °C, pH 7.4 and enzyme triggered release was studied at 37 °C, pH 7.4 in the presence of sPLA2 enzyme. The release profile with multiple triggers was studied at 42 °C, pH 7.4 in the presence of sPLA2 enzyme. 60 ng ml⁻¹ concentration of sPLA2 in the medium was used to study its effect on the release profile.⁸ Dialysis was done by using HIMEDIA® LA 387 Dialysis Membrane-50 (molecular wt. cutoff 5000–10 000) against PBS as release medium in USP dissolution apparatus type-II (Electrolab, TDT-08L). 25% methanol was added to the sink medium to aid solubilisation of the drug in accordance with the method mentioned by Joshi et al.²² The release study was done with a sufficiently good sink condition. Paclitaxel present in the withdrawn aliquots was quantified by UV-spectrophotometry (Perkin Elmer Lambda 25) at 228 nm after appropriate dilutions. In order to understand the effects of individual triggers on the size of the nanovesicles, their size distribution was evaluated at the end of the release study by dynamic light scattering (DLS) using a laser particle analyzer (BI 200SM, Brookhaven Instruments Corporation, USA) and was compared to the original size distribution.

Surface activity measurements

Surface activity measurements for LN-PTX, LN-B and Taxol® were done using a Langmuir–Blodgett instrument (KSV Instrument Ltd., Finland) in order to understand their adsorption patterns at the pulmonary air–aqueous interface. The average surface tension values achieved within 1 second were used as a parameter as described elsewhere,²⁴ as it is desired by an active lung surfactant to rapidly adsorb at an air–liquid interface. The ability of the nanovesicles to reduce the surface tension was suggestive of their ability to mimic the functions of endogenous pulmonary surfactants. The adsorption rate is a crucial parameter and is indicative of the ability of the surfactant to adsorb from the pulmonary alveolar subphase to the air–aqueous interface and also to replenish the interfacial film if matter has been lost or inactivated. The adsorption was studied for 30 minutes for each sample. All the experiments were done in triplicates to ensure reproducibility.

Airway patency

A pulmonary surfactant plays a crucial role in maintaining the patency of narrow airways in the lungs. In case the surface activity of this surfactant is inhibited, the fluid film lining the epithelium of the airways starts moving from wider to narrower airways forming liquid columns that block the airway,²⁵ resulting in the occlusion of terminal airways thereby increasing the resistance to airflow. An efficient pulmonary surfactant on the other hand prevents the occlusion of terminal airways and hence maintains good airway patency. Since LN-PTX is intended for aerosol administration, it is crucial to evaluate its surfactant activity and hence the airway patency. Capillary surfactometer (CS) from Calmia Biomedicals (Toronto, Ontario) was used to study the airway patency and surfactant ability of LN-PTX.⁷ The airway patency of LN-PTX was studied as the % opening time of capillary over an observation period of 120 seconds and was compared against to that of Taxol®.

In vitro lung deposition

In vitro lung deposition studies for LN-PTX and Taxol® were performed using a glass twin impinger apparatus (Copley Scientific, Nottingham, UK), adapted from apparatus A of European and British Pharmacopoeia.²⁶ LN-PTX/Taxol® (1 mg PTX ml⁻¹, 7 ml) suspension was placed in the sample holder and aerosolized using Omron Micron AIR U22 Ultrasonic Nebulizer. A 60 l min⁻¹ air flow rate was maintained inside the impinger during nebulization by a vacuum pump. In the glass twin impinger, 7 and 30 ml of acidified methanol (200 μl of glacial acetic acid to 1 l methanol) was taken in upper and lower chambers, respectively, as per European and British Pharmacopoeia. Nebulization was done for 1 minute and at the end, samples from throat, upper stage (stage I) and lower stage (stage II) were collected and quantified for paclitaxel using HPLC (Agilent 1100 Binary LC pump liquid chromatograph, Zorbax SB C-18 column, 250 × 4.6 mm, 5 μm). The mobile phase was acetonitrile-water (60 : 40 v/v) and the column temperature was maintained at 25 °C. The analysis was performed at a flow rate of 1.5 ml min⁻¹ with a UV detector at 227 nm.

Cell culture

A human non-small cell lung carcinoma (A549) cell line was purchased from National Centre for Cell Sciences (NCCS) Pune, India and was cultured in a 25 cm² tissue culture flask (NUNC, USA) containing DMEM supplemented with 10% FBS and 1% antibiotic antimycotic solution. The cell line was incubated under saturated humid conditions at 37 °C and 5% CO₂. Medium was changed every other day with subculture or plating at around 80% confluency.

In vitro cytotoxicity

In vitro cytotoxicity was evaluated for LN-PTX, LN-B and Taxol® in A549 cells. Around 80% confluent cells were harvested and seeded onto 96 well tissue culture plates at a density of 10⁴ cells per well and incubated for 24 h in saturated humid conditions at 5% CO₂ and 37 °C. Spent medium was then replaced by fresh medium containing formulations with graded concentration of paclitaxel ranging from 10–10 000 nM and plates were further incubated for 72 h. Cells treated with medium only served as control. In order to understand if the trigger mechanisms had an effect on therapeutic efficacy of nanovesicles, the cytotoxicity of LN-PTX was also evaluated by incubating LN-PTX treated cells in the presence of different triggers individually, i.e. 42 °C temperature and sPLA2 enzyme (60 ng ml⁻¹) in the release medium. In order to further understand if the combinatorial application of these triggers had an advantage over single trigger application, cytotoxicity of LN-PTX was evaluated in the presence of both triggers. At the end, SRB (Sulforhodamine B) assay was conducted.²² Cell viability was measured using the formula:

% Viability = Absorbance of sample/Absorbance of control × 100.

IC₅₀ values for all the formulations were calculated using GraphPad Prism 4 software.

Cellular uptake and its mechanism

Cellular uptake of the nanovesicles by A549 cells was studied at three different time points viz. 0.5 h, 1 h and 3 h, by incubating cells with rhodamine-6G (Rh-6G) loaded nanovesicles. Cells were observed using a confocal laser scanning microscope (CLSM) (Olympus Fluoview, FV500, Tokyo, Japan) using an excitation wavelength of 570 nm and an emission wavelength of 590 nm for rhodamine 6G and images were acquired and analyzed with 60× water immersion objective using the Fluoview software (Olympus, Tokyo, Japan). In order to understand the mechanism of cellular uptake, cells were incubated in the presence of Rh-6G loaded nanovesicles under normal and ATP depleted conditions. ATP depleted conditions were obtained by pre incubation of cells in the presence of a metabolic inhibitor, i.e. 0.1% sodium azide and incubation at 4 °C temperature.²⁷ Cellular Rh-6G content was quantified by a fluorescence plate reader (Victor 3V Multilabel Plate Reader, PerkinElmer, USA) (λ_ex = 570 nm and λ_em = 590 nm) and was normalized with respect to the cellular protein content as determined by a Pierce BCA protein assay kit (Thermo Scientific, Pierce, USA). Cellular uptake and its mechanism were further confirmed by flow cytometry analysis of cells incubated for 1 h with Rh-6G loaded nanovesicles and free Rh-6G under normal and ATP depleted conditions. Flow cytometry was done using a BD FACSAria (SOS) flow cytometer (BD Biosciences, USA) and analysis was done by FCS Express software.

Apoptosis assay

A549 cells treated with LN-PTX, LN-B and Taxol® were assayed for apoptosis by studying DNA fragmentation using agarose gel electrophoresis.²⁸ Cells were incubated for 48 h in the presence of LN-PTX or LN-B or Taxol® at equivalent paclitaxel concentration of 1 μM. Cells incubated with medium alone were used as control. The assay for LN-PTX was conducted in the presence of both the triggers, i.e. 42 °C temperature and sPLA2 enzyme (60 ng ml⁻¹) simultaneously, whereas no trigger conditions were used in the case of LN-B and Taxol®. DNA was extracted by a phenol chloroform method and electrophoresis was done at 50 V using 2% agarose gel. DNA was imaged by staining with ethidium bromide using a Gel Doc instrument (Uvi Tech).

Statistics

All the studies were done in triplicates and the results expressed as mean ± standard deviation. Statistical significance of the data was analyzed by Student’s t-test. In all the cases p < 0.05 was considered to be significant.

Results

Both blank and paclitaxel loaded lipid nanovesicles were prepared by modified thin film hydration followed by sonication and extrusion.²² Physiochemical properties of LN-PTX are summarized in Fig. 1. Size distribution of the nanovesicles was found to be unimodal (Fig. 1A) with an average hydrodynamic diameter of 119.8 ± 38.5 nm and a polydispersity index of 0.28 ± 0.05 as determined by DLS. Size of the nanovesicles as obtained by DLS was further supported by transmission electron microscopy (TEM) images as shown in Fig. 1C, which showed 100–200 nm sized, uniformly distributed nanovesicles. Zeta potential as determined by a zeta analyzer was observed to be 4.5 ± 1.5 mV (Fig. 1B). Paclitaxel was encapsulated into the nanovesicles with a high encapsulation efficiency of 82.2 ± 4% as determined by HPLC (Fig. 1B). High encapsulation efficiency of paclitaxel is desirable for formulation development and is a result of highly hydrophobic nature of the drug due to which it has a high affinity for the lipid bilayer of the nanovesicle.

The in vitro release profile of paclitaxel from LN-PTX was studied under normal physiological conditions and was compared to the release profile observed in the presence of single or multiple triggers. The release pattern under normal physiological condition was found to be sustained with 17.4 ± 0.84% cumulative release observed in 72 h (Fig. 2A). This is due to the strong hydrophobic nature of paclitaxel because of which it is strongly bound to the lipid bilayer of the nanovesicle and gets released very slowly into the surrounding medium. During the entire release process, no precipitation or flocculation was observed by visual inspection in the release medium. A statistically significant increase (p < 0.05) in the release was observed in the case of both the triggers used individually. Quantitatively, 26.4 ± 0.81% and 31.6 ± 1.5% cumulative releases were observed in 72 h with temperature trigger of 42 °C and enzyme (EsPLA2, 60 ng ml⁻¹) trigger, respectively, which corresponds to a 1.5 fold and 1.8 fold increase as compared to the release observed under physiological conditions (Fig. 2B). A further increase of 2.4 folds in 72 h with 41.1 ± 0.63% cumulative release was observed in the presence of sPLA2 and 42 °C temperature simultaneously, suggesting the advantage of multiple triggers over single. As evident from the DLS analysis of the nanovesicles at the end of the in vitro release study, i.e. 72 h (Fig. S1, ESI†), the enzyme (sPLA2) trigger significantly decreased the average hydrodynamic diameter of the nanovesicles. Temperature trigger (42 °C) on the other hand did not show any reduction in the size of the nanovesicles (Fig. S1, ESI†).

In the process of assessing the feasibility of nanovesicles for aerosol therapy, their surface activity was evaluated by studying the adsorption profiles. Adsorption profiles of LN-PTX, LN-B and Taxol® were evaluated for 30 minutes using a Langmuir–Blodgett instrument. Over the entire observation period of 30 minutes, adsorption profiles of LN-PTX and LN-B were found to be significantly different (p < 0.05) from that of Taxol® with LN-PTX and LN-B exhibiting lower surface tension values as compared to Taxol® (Fig. 3A). The adsorption profile for LN-PTX however started with a surface tension value comparable to that of LN-B, but showed a marked decrease in the surface tension values with time resulting in a significantly (p < 0.05) lower surface tension value after 30 minutes as compared to that of LN-B. The average surface tension within 1 second for Taxol, LN-B and LN-PTX were 36.46 ± 1.07 mN m⁻¹, 31.8 ± 1.22 mN m⁻¹ and 32.73 ± 0.59 mN m⁻¹, respectively, which finally reached to 36.23 ± 0.44 mN m⁻¹, 26.46 ± 0.95 mN m⁻¹ and 23.25 ± 1.16 mN m⁻¹, respectively, after 30 minutes. On the basis of average surface tension values, LN-PTX was found to be more surface active and hence having a better adsorption profile at the air–aqueous interface as compared to Taxol® and LN-B. The ability of the nanovesicles to mimic the functions of endogenous pulmonary surfactants was also evaluated by studying terminal airway patency in a Capillary Surfactometer. Airway patency of LN-PTX and Taxol® was compared in terms of the % opening time of capillary. LN-PTX showed 99.16 ± 0.5% capillary opening time as compared to the standard paclitaxel formulation (Taxol®) which exhibited 5 ± 1.2% capillary opening time (p < 0.05) for the entire observation period, i.e. 120 seconds (Fig. 3B). Moreover, 99.2 ± 0.3% capillary opening time was observed with LN-B suggesting that the effects observed in the case of LN-PTX are entirely due to the nanovesicle and not because of paclitaxel. This clearly indicates that unlike Taxol®, which has a tendency to block the capillary suggesting its unfavorable effects on airway resistance, nanovesicle based formulation has good surfactant properties, which make it suitable for use in aerosol administration. This will allow for the opening of the upper airways and will help in permeation of aerosol in the narrow passages of the diseased lungs.

In vitro lung deposition of LN-PTX was studied using a glass twin impinger. The amount of drug deposited in the upper impingement chamber (stage I) can be correlated to the drug deposition in the tracheobronchial region whereas the depositions in lower impingement chamber (stage II) correlates to the deposition in the alveolar region or terminal airways. Statistically significant difference (p < 0.05) in deposition was observed between stage II and other stages of the impinger, with deposition in stage II to be significantly (p < 0.05) higher as compared to other stages (Fig. 4). Quantitatively, 86.4 ± 3.2% paclitaxel was deposited in stage II as a result of 1 minute nebulization of paclitaxel. Since, the cutoff aerodynamic diameter for deposition in lower impingement chamber of twin impinger is 6.4 μm at an airflow rate of 60 l min⁻¹ and maximum deposition for LN-PTX was observed to be in stage II, it can be inferred that the mass median aerodynamic diameter (MMAD) of LN-PTX aerosol droplets is below 6.4 μm thereby making it capable of reaching the terminal airways and hence suitable for aerosol drug delivery. Taxol® on the other hand showed less than 8% and similar depositions in all the three stages indicating its inability for aerosol administration.

Fig. 1 Physiochemical characterization of LN-PTX. (A) Size distribution of LN-PTX obtained using dynamic light scattering (DLS) shows unimodal characteristics. (B) Encapsulation efficiency of paclitaxel in LN-PTX and their zeta potential. (C) Transmission electron microscopy (TEM) image of LN-PTX. Scale bar: 200 nm.

Fig. 2 (A) Release of paclitaxel from LN-PTX under different conditions with pH maintained at 7.4. (B) Folds increase in 72 h cumulative release in response to trigger (42 °C temperature or 60 ng ml⁻¹ sPLA2 individually or combined) as compared to release obtained under normal conditions. * indicates statistically significant difference among different groups (p < 0.05).

Fig. 3 (A) Adsorption profile of LN-PTX compared with LN-B and Taxol®. (B) Percentage opening time of capillary as measured with a capillary surfactometer.

Fig. 4 Percentage deposition of paclitaxel in different stages of twin impinger as a result of 1 minute nebulization of LN-PTX and Taxol®.

After understanding the advantage of multi trigger responsive nanovesicles towards controlled release of paclitaxel and evaluating their pulmonary surfactant mimetic nature required for aerosol therapy, we tried to understand if the use of multiple triggers poses a benefit in terms of improved therapeutic efficacy. For this, we evaluated the cytotoxicity of different formulations in human non-small cell lung carcinoma (A549) cells by SRB (Sulforhodamine B) assay over a period of 72 h of incubation. All the formulations but LN-B exhibited a dose dependent cytotoxic effect. LN-B did not exhibit any cytotoxicity even at concentrations as high as 100 μM suggesting that the nanovesicle itself did not contribute to the cytotoxicity observed with LN-PTX. Fig. 5 shows the IC₅₀ values for LN-PTX and Taxol® under different conditions. IC₅₀ values for LN-PTX (no trigger) and Taxol® were found to be 106 ± 17 nM and 149 ± 3 nM respectively. A statistically significant decrease (p < 0.05) in the IC₅₀ value in the case of LN-PTX indicates the clear advantage of nanovesicles towards increasing the therapeutic efficacy of paclitaxel. With the use of single triggers, IC₅₀ values for LN-PTX were further lowered to 84 ± 5 nM and 78 ± 4 nM in the case of temperature trigger and sPLA2 trigger, respectively, which suggests the improved cytotoxic potential of nanovesicles in response to the individual trigger mechanisms. Finally, statistically significant reduction (p < 0.05) in the IC₅₀ value of LN-PTX to 49 ± 2 nM was observed when both the triggers, i.e. temperature and sPLA2, were used simultaneously. These results suggest the advantage of multi trigger responsive nanovesicles over single trigger responsive nanovesicles in terms of increased therapeutic efficacy.

We also tried to understand the cellular uptake of nanovesicles by A549 cells which was done by incubating cells with Rh-6G loaded nanovesicles for different time points. As shown in Fig. 6A, cells incubated in the presence of Rh-6G loaded nanovesicles showed bright fluorescence, co-localized uniformly inside the cells at all time points. In contrast to this, cells incubated with free Rh-6G showed negligible fluorescence (data not shown). This finding implies the role of these nanovesicles in mediating and facilitating the cellular uptake of the encapsulated material. In order to further confirm the cellular internalization of the nanovesicles and eliminate the probability of any superficial fluorescence as a result of nanovesicles localized on the surface of the cells, Z scan or depth scan of cells was done. The Z scan for a 3 h time point, as shown in Fig. 6B, was done over a range of −10 μm to +10 μm and showed highest fluorescence intensity near to 0 μm. This confirmed that the nanovesicles were completely internalized by the cells and were not present at the surface. In the process of understanding the mechanism of cellular uptake of these nanovesicles, it was observed that cells pretreated with 0.1% sodium azide and cells incubated at 4 °C showed significantly less (p < 0.05) intracellular Rh-6G content at all time points as compared to those incubated under normal conditions, i.e. 37 °C without azide (Fig. 6C). Sodium azide being a metabolic inhibitor depletes the cell of ATP and hence no active process is possible thereafter. Similar effect is also caused by the incubation of cells at 4 °C rather than 37 °C. This suggests that the cellular uptake of the nanovesicles is an energy dependent or active process. Flow cytometry analysis of cells incubated for 1 h further confirmed these findings, as a significant decrease (p < 0.05) in both percentage positive cells and mean fluorescence intensity was observed in the case of cells incubated with Rh-6G loaded nanovesicles under ATP depleted conditions as compared to cells incubated under normal conditions (Fig. S2, ESI†), which indicates ATP mediated cellular uptake of the nanovesicles. Also, cells incubated with free Rh-6G showed significantly less (p < 0.05) percentage positive cells and significantly less (p < 0.05) mean fluorescence intensity as compared to cells incubated with Rh-6G loaded nanovesicles (Fig. S2, ESI†), indicating facilitated endocytosis of the nanovesicles. Chromatin condensation, DNA fragmentation followed by eventual degradation of DNA into small apoptotic bodies are important events involved in the apoptosis or programmed cell death.²⁹ Apoptosis assay for the cells treated with LN-PTX, LN-B and Taxol® was done by studying DNA fragmentation using 2% agarose gel electrophoresis. Treatment with LN-PTX was done in the presence of both temperature and sPLA2 triggers simultaneously. As can be seen in Fig. 7, DNA smear was obtained for the cells treated with LN-PTX and Taxol®. Control cells (without any treatment) did not show any visible DNA fragmentation. The DNA ladder/smear observed in the case of Taxol® treated and LN-PTX treated cells is in accordance with literature evidence which suggests that paclitaxel induces programmed cell death in human non-small cell lung carcinoma cells by increasing caspase-3 activity and causing DNA fragmentation.³⁰

Fig. 5 72 h IC₅₀ values for different formulations under different conditions in A549 cells. * indicates statistically significant difference of LN-PTX (Temp + sPLA2) IC₅₀ as compared to other groups.

Fig. 6 (A) CLSM images of A549 cells after incubation with Rh-6G loaded nanovesicles for different time points. (B) CLSM images of cells incubated with Rh-6G loaded nanovesicles for 3 h in Z scan mode with the scanning done from −10 μm to +10 μm. (C) Cellular levels of Rh-6G after incubation of A549 cells with Rh-6G loaded nanovesicles under normal and ATP depleted conditions.

Fig. 7 DNA fragmentation study of A549 cells treated with different formulations at 1 μM paclitaxel concentration for 48 h.

Discussion

Disadvantages associated with conventional chemotherapy for cancer has resulted in the research and development of several kinds of drug delivery systems over the last three decades. Lipid based nanoparticles such as liposomes or lipid nanovesicles and solid lipid nanoparticles (SLN) have been extensively studied as the carriers for various antineoplastic agents.^1,31,32 In the present study lipid nanovesicles have been optimized for their temperature, enzyme sensitivity to be used as multiple triggers and their surface activity to mimic pulmonary surfactant function. In this regard, we developed paclitaxel loaded lipid nanovesicles sensitive to both temperature (42 °C) and sPLA2 enzyme (a tumor specific enzyme), evaluated them towards feasibility for aerosol administration and advantages of using multiple triggers as compared to single in terms of therapeutic efficacy of the system.

Size distribution for the nanovesicles (LN-PTX) was found to be unimodal and uniform with size range from 100–150 nm, which is desirable as they can reach the terminal regions of the lung due to a lower MMAD and can avoid unwanted mucociliary clearance.^33,34 A near to neutral zeta potential of LN-PTX is not in support with their stability in suspension. However, the nanovesicles can be stable because of their high surface activity. The high encapsulation efficiency of paclitaxel (∼81%) is attributed to its hydrophobic nature and hence a high affinity for the lipid bilayer which also resulted in a slow and sustained release of paclitaxel from LN-PTX under physiological conditions, which is desirable. A significant increase in the release of paclitaxel was observed in the presence of trigger conditions. An increase in temperature from 37 °C to 42 °C resulted in gel-to-liquid phase transition of the lipid bilayer. This can have implications in adjuvant therapies like hyperthermia and photothermal therapy wherein the heat generated as a result of externally applied alternating magnetic field or light can increase the local temperature of the tumor region to just above the physiological temperature resulting in the triggered release of the encapsulated drug.^10,35 The presence of sPLA2 enzyme in the release medium results in the hydrolysis of the phospholipids resulting in the generation of free fatty acids and lysophospholipids which can further enhance the permeability of the lipid bilayer thereby resulting in triggered release of paclitaxel.¹¹ A statistically significant decrease in the size of the nanovesicles with the use of enzyme (sPLA2) trigger suggests degradation of the nanovesicles by sPLA2, which eventually results in triggered release of the drug. sPLA2 mediated degradation of nanovesicles can also have implication towards their improved penetration through a dense interstitial tumor matrix due to their improved diffusivity, as reported by Wong et al.³⁶ With temperature trigger (42 °C), the nanovesicles though did not show any reduction in size after 72 h, but exhibited significant response in terms of triggered release of the drug. This is due to the fact that unlike enzyme (sPLA2) which triggers the release from nanovesicles by degrading them, an increase in temperature triggers the release by causing phase transition of the lipids and not affecting their size. Use of both the triggers simultaneously resulted in further increase in the cumulative release of paclitaxel indicating the advantage of a multi trigger responsive system over single trigger responsive ones.

Lipid nanovesicles have been previously explored for aerosol therapy in lung cancer.^1,37 As much as their advantage in terms of improved antitumor efficacy has been looked upon, their optimization in relation to surface activity to make them more similar to natural pulmonary surfactants has not been given much attention. In this work, we took this issue into account and evaluated the nanovesicles for their endogenous pulmonary surfactant mimetic properties. LN-PTX showed significantly improved adsorption and surface activity as compared to Taxol®, which could be due to surface active lipid nanovesicles used as the carriers for paclitaxel. DSPC, the major component of LN-PTX and a naturally occurring endogenous lung surfactant, will alone adsorb poorly at the air–water interface at temperature below its gel to liquid transition temperature (∼56 °C), as is the reported case with another saturated phospholipid, i.e. DPPC.⁷ The combination of DSPC with POPE, an unsaturated phospholipid which is also one of the components of an endogenous lung surfactant, showed a better adsorption profile and surface activity. This can be attributed to the unsaturated nature of POPE, addition of which to DSPC lowered down the gel to liquid transition temperature of the lipid mixture as compared to that of DSPC resulting in rapid opening of the nanovesicles at physiological temperature and forming a monolayer at the air–aqueous interface. This is in accordance with one of our previously reported findings wherein the use of an unsaturated phospholipid with DPPC showed improved adsorption profile.²² Surface tension values within 1 second were similar for LN-PTX and LN-B. However, a marked decrease in the surface tension value of LN-PTX was observed with time and this decrease was found to be significantly more as compared to the decrease observed in the case of LN-B. The improved adsorption of LN-PTX compared to LN-B with time may be attributed to the miscibility of the two components (lipids and paclitaxel) which form a mixed monolayer at the air–aqueous interface after the opening of the nanovesicles as documented by Feng et al.³⁸ using a Langmuir monolayer of phospholipids and paclitaxel. Paclitaxel released from the nanovesicles, upon their rapid opening at the air–aqueous interface, forms a mixed lipid monolayer thereby increasing the mobility of the acyl chains of the lipid, resulting in the fluidizing effect on the monolayer and reorganization of their microdomains.³⁷ In the process of adsorption, the nanovesicles transport from subphase to the interphase followed by conversion from bilayer structures to interfacial layers.³⁹ Rapid adsorption of LN-PTX nanovesicles would therefore cause the release of paclitaxel at the air–liquid interface due to opening of the nanovesicles, thereby acting as a drug-delivery vehicle. Also, paclitaxel in the form of Taxol® does not exhibit good surface activity in spite of the presence of Cremophor® EL, a surfactant, in the formulation. Poor surface activity of Taxol® can be attributed to the presence of dehydrated ethanol in the formulation. The surface active nature of LN-PTX will allow for the opening of the upper airways resulting in their easy permeation in the narrow passages of the diseased lungs when given as aerosol. This was confirmed by studying the airway patency using Capillary Surfactometer. Taxol®, due to the poor surface activity as observed in adsorption studies, showed poor airway patency. Even Abraxane®, the albumin nanoparticle based commercial formulation of paclitaxel cannot meet these challenges of achieving good surface activity and airway patency due to the presence of albumin in it, which is a known inhibitor of endogenous lung surfactant activity.⁴⁰ Maximum deposition of paclitaxel was obtained in the lower impingement chamber of a twin impinger following a 1 minute nebulization of LN-PTX suggesting that the MMAD of most of the aerosol droplets of LN-PTX was well below 6.4 μm, further suggesting it to be capable of reaching the terminal regions of the lung.²⁶

Cell cytotoxicity study in A549 cells showed significantly less IC₅₀ for LN-PTX as compared to Taxol, suggesting the advantage of nanovesicle based formulation over marketed formulation of paclitaxel. The IC₅₀ values were further lowered in the presence of trigger conditions with minimum IC₅₀ observed in the case of multiple triggers used simultaneously. Use of trigger stimulus resulted in a significant increase in the release of paclitaxel from LN-PTX thereby preventing the lysosomal degradation of the drug upon cellular uptake which is otherwise the case. Also, in the case of sPLA2 used as the trigger mechanism, the lysophospholipids released due to the hydrolysis of phospholipids by sPLA2 enzyme act as the permeability enhancers thereby making the plasma membrane of cells more permeable so that the released drug guides its way into the cells. Increased cytotoxicity observed with simultaneous use of multiple triggers is in accordance with in vitro release data which showed maximum release in the presence of both the triggers. There are a few previous reports exploring the advantages of multi trigger responsive systems over single ones.^41,42 However, none of them have focussed on the exploration of multi trigger mechanisms in the case of surface active nanoparticle systems intended for local or regional delivery of drugs as in aerosol.

LN-PTX was found to induce apoptosis in A549 cells by causing DNA fragmentation, a mechanism similar to that of Taxol® or free paclitaxel.³⁰ Therefore the present system improves the therapeutic potential of paclitaxel without changing its mechanism of action. The nanovesicles also facilitated the cellular uptake and interaction of the encapsulated material as observed in the cellular uptake study. We speculate that the improved cellular interaction of the nanovesicles can be due to their surface active nature as there are previous reports wherein it the use of surface active materials has been shown to enhance interaction with biological membranes.^43,44 The endocytosis of the nanovesicles was found to be ATP mediated and hence an active process.

Conclusions

We developed temperature and enzyme sensitive, surface active lipid nanovesicles for aerosol therapy in lung cancer. The nanovesicles exhibited high surface activity and airway patency which make them endogenous pulmonary surfactant mimetic and thereby establish their feasibility for aerosol administration. The use of multiple triggers simultaneously showed a substantial increase in the paclitaxel release leading to the enhanced therapeutic efficacy in human non-small cell lung carcinoma (A549) cells which showed apoptosis as a result of their exposure to the nanovesicles. Due to temperature sensitivity, these nanovesicles can also have implications in adjuvant therapies like hyperthermia and photothermal therapy. The surface active nature of the nanovesicles also resulted in their improved cellular interaction and uptake. In all, these multi trigger responsive, surface active nanovesicles offer a promising platform for improved delivery and efficacy of paclitaxel in lung cancer.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Details of experimental methods, Fig. S1 and S2. See DOI: 10.1039/c2ib20122d

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