Lipid-PLGA hybrid nanoparticles of paclitaxel: Preparation, characterization, in vitro and in vivo evaluation

Journal Pre-proof Lipid-PLGA hybrid nanoparticles of paclitaxel: Preparation, characterization, in vitro and in vivo evaluation

Sandeep Godara, Viney Lather, S.V. Kirthanashri, Rajendra Awasthi, Deepti Pandita PII:

S0928-4931(19)34064-0

DOI:

https://doi.org/10.1016/j.msec.2019.110576

Reference:

MSC 110576

To appear in:

Materials Science & Engineering C

Received date:

1 November 2019

Revised date:

12 December 2019

Accepted date:

19 December 2019

Please cite this article as: S. Godara, V. Lather, S.V. Kirthanashri, et al., Lipid-PLGA hybrid nanoparticles of paclitaxel: Preparation, characterization, in vitro and in vivo evaluation, Materials Science & Engineering C (2018), https://doi.org/10.1016/ j.msec.2019.110576

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© 2018 Published by Elsevier.

Journal Pre-proof Lipid-PLGA Hybrid Nanoparticles of Paclitaxel: Preparation, Characterization, In Vitro and In Vivo Evaluation Sandeep Godaraa, Viney Latherb, S. V. Kirthanashric, Rajendra Awasthib, Deepti Panditac* a

Department of Pharmaceutics, Jan Nayak Ch. Devi Lal Memorial College of Pharmacy,

Sirsa-125055, Haryana, India b

Amity Institute of Pharmacy, Amity University Uttar Pradesh, Sector-125, Noida 201313,

India c

Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), Amity

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University Uttar Pradesh, Sector-125, Noida 201313, India

*

Corresponding author:

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Amity Institute of Molecular Medicine & Stem Cell Research (AIMMSCR), Amity University Uttar Pradesh, Sector-125, Noida 201313, India

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Tel.: +91 9991776090

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E-mail: [email protected], [email protected]

Declaration of interests: The authors declare that they have no conflict of interest.

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Journal Pre-proof ABSTRACT Paclitaxel loaded lipid-polymer nanoparticles (NPs) were successfully synthesized using poly lactide-co-glycolide (PLGA) as polymer and stearyl amine, soya lecithin as lipids via single step nanoprecipitation method. The study was aimed to combine the advantage of structural integrity of hybrid NPs containing PLGA core and lipid in the shell. Surfactants such as polyvinyl alcohol (PVA), tocopheryl polyethylene glycol succinate (TPGS), pluronic 68 (F68) and human serum albumin (HSA) were used as stabilizers. NPs were characterized w.r.t. morphology, particle size, zeta potential, encapsulation efficiency, in vitro drug release, protein binding capability and blood compatibility. NPs were in size range of 150-400 nm

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and the particle size was greatly influenced by type and concentration of surfactants and

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lipids. TEM analysis confirmed the spherical shape and coating of the lipid on the NPs surface. Highest percentage entrapment efficiency was observed in NPs prepared with HSA

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as surfactant. The release rate of paclitaxel from modified NPs was much slower as compared to unmodified NPs. The percent protein binding of P-PVA, P-TPGS, P-F68 and P-HSA

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(unmodified NPs) was found to be 15.11%, 16.27%, 27.90% and 33.72%, respectively demonstrating effect of surface properties of NPs on protein binding. The hemolytic activity

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of the NPs was found to be dependent on type of surfactant and not on the lipid employed. PVA, TPGS, F68, HSA surfactants showed ~16%, ~10%, ~13%, ~7% hemolysis rate,

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respectively. The surface nature of NPs had a significant effect on the circulation profile of formulations. The HSA based NPs showed prolonged blood circulation time when compared

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to NPs without lipid coating. Thus, the synthesized dual lipid coated PLGA NPs with HSA could act as a potential nano-system for controlled delivery of paclitaxel.

Keywords:

Blood

compatibility, lipid-polymer hybrid

pharmacokinetics, PLGA, surface modification

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nanoparticles,

nanoparticles,

Journal Pre-proof 1. INTRODUCTION During the past few decades, the growing interest in the development of nanocarriers such as polymeric nanoparticles (NPs) and lipid NPs has been evidenced from the increasing number of patents and publications [1, 2]. Fluctuation in plasma-drug concentration is the major limitation associated with the conventional NPs. Lipid based NPs possess amphiphilic side chains that can be readily functionalized, are biocompatible and have a longer circulation time. However, instability and structural loss are the major limitations of lipid based NPs. Polymeric NPs are comparatively stable for drug loading due to their structural stability [3]. To circumvent the limitations associated with these NPs, focus is being inclined towards the

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development of modified nanosystem, which produces a combined benefit of polymeric

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material and lipid. Hybrid NPs possess a combination of characteristics as these are consisted of polymeric core and lipid shell. These are stable particles and allow high drug loading [4].

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Additionally, outer lipid coating retards polymer degradation by restraining inward diffusion of water, thus facilitating uniform and continuous release temperament to the incorporated

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substance(s). Hybrid NPs have been reported as a superior candidate for drug delivery by Yu et al., where the delivery of salinomycin was controlled when conjugated in polymer-lipid

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hybrid NPs [5]. Soya lecithin-poly lactide-co-glycolide (PLGA) hybrid NP with Psoralen exhibited excellent inhibitory effect on breast cancer cell line due to the delayed drug release

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from lipid-polymer structure [6]. Salzano et al., reported sustained release of daunorubicin and lornoxicam from hybrid PLGA/ lipid NPs [7]. Hybrid NPs with PLGA core have been

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reported to deliver amphotericin B using stearyl amine to form a cationic shell to target ligand for macrophages cells. These core-shell NPs were stable, had a high encapsulation efficiency and showed slow release of Amphotericin B [8]. Adsorption of apolipoproteins to polysorbate 80 coated NPs enhanced the permeation of NPs across the blood brain barrier [9]. The cellular interaction of NPs with cancer cells can also enhanced by vitronectin adsorption to the NPs [10]. The human blood consists of a huge array of protein molecules that interact with the surface of the NPs [11, 12]. The protein-protein interaction (van der Waals and electrostatic interactions) and the binding affinity of proteins on NPs determine the protein adsorption on its surface. The adsorbed protein opsonins (complement proteins, IgG and laminin) promote phagocytosis via mononuclear phagocyte system that quickly eliminate NPs from the bloodstream, while dysopsonins (human serum albumin (HSA) or apolipoproteins) reduces the phagocytosis and leads to prolonged circulation time in blood [13]. The surface chemistry of NPs (e.g. surface electric charge, surface density) plays a dominant role in the recognition 3

Journal Pre-proof of protein on its surface. Upon interaction with blood, NPs form a protein coat termed as cornea [14]. In order to minimize the protein adsorption, NPs are treated with poly(ethylene glycol) (PEG); the hydrophilic moiety reduces the uptake by reticuloendothelial cells [15]. Increase in plasma binding efficiency and higher circulation time with blood have been reported in PEG and pluronics coated PLGA NPs [16]. Also, lipid based nano carriers exhibit steric repulsion, thus making them potential candidate for drug delivery [17, 18]. Pirarubicin loaded lipid-hybrid albumin nanoparticle containing oleic acid as the lipophilic complexing agent have shown promising antitumor activity [19]. Paclitaxel (a diterpenoid pseudoalkaloid) has been recognized as a significant

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chemotherapeutic. It was isolated from the Pacific Yew bark in early 1960s. It has neoplastic

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activity against breast, colon, primary epithelial ovarian, and non-small cell lung cancers. It is approved for human use in many countries for second line treatment of breast and ovarian

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cancers [20]. Poor water solubility of paclitaxel makes it challenging for its successful delivery to the target cancer cells. The successful example of albumin application in drug

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delivery system is FDA approved AbraxaneTM (albumin-bound paclitaxel nanoparticle), developed by American Bioscience, Inc. for the treatment of metastatic breast cancer.

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The purpose of this communication is to explore polymer-lipid hybrid NPs for controlled delivery and better pharmacokinetic potential of paclitaxel. To achieve this objective,

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paclitaxel loaded polymer-lipid hybrid NPs were synthesized using a single step nanoprecipitation method. The hybrid NPs contained PLGA core and lipid combination

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(stearyl amine and soya lecithin) in the shell. The effect of various surfactants such as polyvinyl alcohol (PVA), pluronic 68 (F-68), tocopheryl polyethylene glycol succinate (TPGS) and human serum albumin (HSA) was also examined. The synthesized NPs were subsequently characterized for size, morphology, surface charge, entrapment efficiency, protein binding, and in vitro drug release. In vitro hemolysis study was carried out to examine blood compatibility. To ascertain the in vivo fate of the orally administered NPs, the pharmacokinetic studies were carried out in male Wistar rats.

2 MATERIALS AND METHOD 2.1 Materials Paclitaxel was received as a gift sample from Fresenius Kabi Oncology Ltd., India. Poly(lactic-co-glycolic acid) (PLGA) 80:20, Mw 50,000-75,000, polyvinyl alcohol (PVA), F68 and human serum albumin (HSA) were purchased from Sigma, USA. Stearyl amine (MW 269.51 g/mol) was purchased from Sigma Aldrich, St Louis, MO, USA. Soya lecithin (95%) 4

Journal Pre-proof was procured from Lipoid, Germany. Tocopheryl polyethylene glycol succinate (TPGS) was obtained from Sigma, France. Dialysis cellulose membrane (MW cut-off 12k Da) was purchased from Sigma Aldrich, USA, and Bradford reagent was obtained from Sigma Life Science, India. The blood samples from a healthy human volunteer were collected at Jan Nayak Ch. Devi Lal Hospital, Sirsa, India. Ethylene di-aminetetraacetic acid (EDTA) was procured from Sisco Research Laboratories Pvt. Ltd., Delhi, India. All the chemicals and solvents, unless otherwise stated, were of analytical grade and purchased from SigmaAldrich.

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2.2 Preparation of paclitaxel loaded PLGA-lipid hybrid NPs

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A single step nanoprecipitation method was used to prepare hybrid NPs [18]. Briefly, the organic phase was formed by dissolving 10 mg paclitaxel, 25 mg lipid and 50 mg PLGA in 3

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mL dichloromethane (DCM). This was added drop-wise to the aqueous phase containing 5 mg/mL of stabilizer and sonicated for 60 seconds, followed by homogenization at 15,000 rpm

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for 20 min using a high speed homogenizer (Heidolph Instruments GmbH and Co. KG, Germany). The obtained nanoemulsion was stirred overnight at the room temperature to

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evaporate DCM. The resulting NP suspension was centrifuged twice at 11,000 rpm (REMI, India) for 10 min to remove the non-encapsulated drug and excess amount of lipid. The

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paclitaxel loaded NPs were collected and lyophilized (Fig. 1). Four different stabilizers namely, PVA, F-68, TPGS and HSA, and two lipids namely, stearyl amine (SA) and soya

properties.

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lecithin (SL) were employed to obtain hybrid NPs with different combinations and surface

Fig. 1: Schematic presentation of albumin coated lipid-PLGA hybrid nanoparticle preparation and drug loading.

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Journal Pre-proof 2.3 Determination of surface morphology The morphological characterization of paclitaxel loaded PLGA-lipid NPs was done by transmission electron microscopy (Morgagni 268D, FEI Co., The Netherlands). A drop of nanoparticle suspension was added to 2% (w/v) aqueous solution of phosphotungstic acid for contrast enhancement and transferred on 400 mesh copper grid and dried before scanning.

2.4 Fourier transfer infra-red (FTIR) spectroscopy The lyophilized hybrid NPs (2 mg each) with and without drug were mixed with potassium bromide (KBr) and transformed into pellets. The spectra were analyzed in the wavelength

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range of 4,000-400 cm−1 by FTIR spectroscopy (Shimadzu Corporation, IR solution

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software).

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2.5 Particle size and zeta potential measurements

The particle size and zeta potential measurements of the synthesized hybrid NPs were carried

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out using Zetasizer (Nano-ZS90, Malvern Instruments, UK). All the determinations were

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done in triplicate and data was presented in mean ± S.D (n = 3).

2.6 Determination of percentage entrapment efficiency (%EE)

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For the determination of %EE, NPs suspension was centrifuged at 10,000 rpm for 20 min and the supernatant was filtered using 0.2 μm syringe tip filter. The amount of paclitaxel in NPs estimated

by HPLC

(LC-2010C

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was

HT, Shimadzu Corporation,

Japan).

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chromatographic separation was accomplished using Luna 5u C18 100A (250 x 4.6 mm) column (phenomenex, USA). The mobile phase consisting of acetonitrile: water (80: 20 v/v) mixture was filtered through 0.45 µm membrane filter, degassed and pumped at a flow rate of 1.0 mL/min with detection wavelength of 228 nm. Analytical data were acquired using LC solution software version 1.25. Sampling was carried out in triplicate. The %EE was calculated using the following formula:

2.7 In vitro drug release study In vitro release profile of paclitaxel from the synthesized hybrid NPs was estimated using dialysis bag technique over a period of 120 h. Briefly, the dialysis bag was immersed in phosphate buffered saline (PBS), pH 7.4 for 5 min. The swelled bags were sealed on one end 6

Journal Pre-proof and the hybrid NPs (2 mL equivalent to 0.5 mg of drug) were poured from the other end into the bag and sealed. Further, the bag was immersed in 20 mL PBS (pH 7.4) containing 5% w/v Tween 80 and 20% methanol, magnetically stirred at 200 rpm. The temperature was maintained at 37 ± 0.5°C. Dialyzing medium (1 mL) was collected at predetermined time intervals, followed by immediate replacement with fresh PBS (pH 7.4) to maintain sink condition and the drug was analyzed by HPLC [21]. The sampling was done in triplicate. To know the precise drug release kinetics from the prepared hybrid NPs release kinetics models viz. zero order and first order models were applied on the release data. The drug release

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mechanism was confirmed using Higuchi’s and Korsemeyer-Peppas’ equations.

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2.8 Plasma protein binding study

Protein binding of paclitaxel from the hybrid NPs was analyzed using a previously reported

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method [22]. Briefly, the blood sample collected from a healthy volunteer was centrifuged at 3000 rpm for 20 min to collect plasma component and stored at -20 ºC. About, 100 μL of the

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plasma sample was taken in Eppendorf tubes containing hybrid NPs suspension to a total volume of 500 μL and vortexed for 10 min. Incubation of the plasma-hybrid NPs suspension

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was carried out at 25 ºC for 2 h. This was followed by the centrifugation at 12, 000 rpm for 10 min. The obtained NP pellets were washed with 600 μL McIllvaine’s buffer (0.1 M di

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sodium phosphate and 0.2 M citric acid) at pH 7.5 to remove unbound protein and centrifuged again. The unbound protein fraction was calculated by mixing washing and

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original supernatants and analyzing by Bradford assay method.

2.9 In vitro plasma stability study

In vitro plasma stability study was performed to assess the plasma enzyme induced degradation of hybrid NPs using a slight modification of the previously reported method [23]. Briefly, a mixture of 900 μL hybrid NPs and 100 μL human plasma sample was placed in a dialysis bag and subjected to in vitro drug release study as described above. The study was performed in triplicates. The mixture of drug and plasma served as control.

2.10 In vitro hemolysis study In vitro hemolysis studies were carried out to understand the blood compatibility profile of the synthesized NPs. The method involved spectrophotometric determination of haemoglobin. Hybrid NPs were incubated with blood and undamaged cells and separated by centrifugation [24, 25]. Briefly, the blood samples were collected from healthy volunteers 7

Journal Pre-proof and stored in EDTA containing tubes. The red blood cells (RBCs) were separated by centrifugation at 3000 rpm for 20 min and erythrocytes were washed repeatedly with PBS (pH 7.4). Three milliliter centrifuged erythrocytes were mixed in 11 mL of PBS to make stock dispersion. Then 100 μL RBC suspension was added in 1 mL of hybrid NPs suspension and incubated at 37 ºC for 0.5 h to 1 h. Further, the supernatant was centrifuged at 3500 rpm for 5 min with PBS (pH 7.4) and analyzed at 540 nm. Distilled water was used as positive control (100% lysis) and saline solution as negative control (0% lysis).

2.11 Pharmacokinetic study

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Pharmacokinetic studies were performed in male Wistar rats (150-200 g), procured from Lala

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Lajpat Rai University of Veterinary and Animal Sciences, Hisar, India. The experimental protocol followed guidelines of the Committee for the Purpose of Control and Supervision of

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Experiments on Animals (CPCSEA), Delhi, India. The protocol was approved by the Institutional Animal Ethics Committee (IAEC) of Jan Nayak Ch. Devi Lal Memorial College

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of Pharmacy, India (JCDMCOP/IAEC/06/16/33). Animals were kept in cages under controlled conditions (i.e. 20 ± 2 °C temperature at 50 ± 2% relative humidity) and

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accommodated to the housing environment for 1 week before the experiment. The rats were randomly divided into eight groups and received treatments as: group 1:

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paclitaxel suspension, group 2: P-PVA, group 3: P-TPGS, group 4: P-F68, group 5: P-HSA, group 6: SA-P-HSA, group 7: SL-P-HSA, and group 8: SA/SL-P-HSA, administered orally

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in a of dose 10 mg/kg body weight. Blood samples were collected in heparinized capillary tubes from the retro-orbital sinus under mild anaesthesia at predetermined intervals. The plasma was separated via centrifugation at 3000 rpm for 10 min. The samples were labelled and stored at -20 ºC till further analysis.

3. RESULTS AND DISCUSSION 3.1 Preparation and characterization of paclitaxel loaded PLGA-lipid hybrid NPs PLGA-lipid hybrid NPs were synthesized by nanoprecipitation approach. The NP core consisted of PLGA and shell consisted of lipid. Homogenization was employed, which uses low ultrasonic vibrational energy for atomization that releases high energy, to form nano-size droplets leading to the synthesis of NPs [26, 27]. In order to optimize the NPs formulation, lipid part was varied, utilizing SA and SL alone and/ or in combination. Additionally, to select an adequate stabilizer which could effectively ensure the stability of hybrid NPs, the

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Journal Pre-proof influence of different stabilizers i.e. PVA, TPGS, F-68 and HSA on the physicochemical properties of the NPs was investigated.

The particle size of NPs decreased in the order of PVA > F-68 > TPGS > HSA for the formulations using either SA or SL (Table 1). The NPs containing PVA had highest average particle size due to the formation of strong hydrogen bonds via hydroxyl group between intra or inter-molecules of PVA, and the gelatinization of PVA at the oil/ water interface [28]. The disulfides bridges present in HSA can prevent agglomeration and stabilized the NPs. Furthermore, the aggregation of drug particles can be prevented by TPGS due to the steric

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effect, and thus, stabilizes the NPs. Furthermore, the entrapment efficiency of paclitaxel

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loaded lipid-PLGA hybrid NPs increased in the order of F-68 < TPGS < PVA < HSA and TPGS < F-68 < PVA < HSA for the formulations employing SL and SA, respectively. The

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hydrophilic side chains on the surface of HSA facilitated an increase in the entrapment efficiency of paclitaxel [29]. These results demonstrated smallest particle size and relatively

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higher percentage drug entrapment in case of hybrid NPs stabilized with HSA.

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The lipids are complex molecule with longer chain and higher viscosity that contribute to the increased NPs size. However, this property of lipid plays a vital role to achieve higher

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encapsulation efficiency. The combination of lipids resulted in higher particles size and increased encapsulation efficiency. The trend observed in respect of increased particle size

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and percentage entrapment efficiency for the surfactants used with the dual lipid i.e. SA and SL coating. In the present study, it was observed as HSA > TPGS > F-68 > PVA and PVA > HSA > TPGS > F-68, respectively.

The zeta potential value plays a crucial role in the physical stability of NPs. Zeta potential value indicates the degree of repulsion between similarly charged particles, which is a predictive test to ensure long term stability of the NPs. In the present study, the zeta potential of P-PVA, P-TPGS, P-F68 and P-HSA formulation was recorded as -32.7 mV, -24.2 mV, 11.8 mV and -26.2 mV, respectively (Table 1). SL coated NPs showed a negative shift in the zeta potential (-22 mV to -35 mV), while the SA coated NPs showed positive zeta potential (28 to 38 mV), which is due to the electrostatic interaction that impacts the cationic charges to the NPs [28]. SL coated NPs were negatively charged, whereas when SA (positively charged lipid) was incorporated into SL vesicles, cationic NPs were obtained due to electrostatic interactions between positively charged lipid (SA) and negative charged lipid 9

Journal Pre-proof (SL). Negative charge of SL is due to the presence of phosphatidic acid [30]. The synthesized NPs containing a combination of SA and SL in the lipid phase had positive zeta potential values. Similar effect of lipid combination of the zeta potential results have been reported previously [31].

Table 1: Composition of different batches of synthesized hybrid nanoparticles. Formulation

Amount of material used (mg) Lipid (mg)

potential

EE%

(mV)

(5 mg/ml)

P-PVA

-

-

PVA

355±43

-32.7±3.4

47.50±1.4

SA-P-PVA

25

-

PVA

380±25

38.2±2.5

59.34±3.5

SL-P-PVA

-

25

PVA

376±68

-35.3±1.4

62.07±2.9

SA/SL-P-PVA

12.5

12.5

PVA

397±34

24.6±0.6

69.47±5.4

P-TPGS

-

-

TPGS

227±56

-24.2±3.5

37.23±3.5

SA-P-TPGS

25

-

TPGS

253±41

30.5±1.9

42.72±2.4

SL-P-TPGS

-

25

TPGS

246±34

-27.1±1.3

45.83±2.8

SA/SL-P-TPGS

12.5

TPGS

268±38

22.3±0.8

62.27±4.3

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P-F68

12.5

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SL

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SA

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Size (nm)

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Surfactant

Zeta

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code

Particle

-

-

F68

261±21

-11.8±2.1

42.39±4.6

25

-

F68

285±56

28.7±1.5

46.11±2.8

SL-P-F68

-

25

F68

272±38

-22.4±1.9

43.56±3.6

SA/SL-P-F68

12.5

12.5

F68

298±22

28.9±0.5

58.61±0.6

P-HSA

-

-

HSA

158±17

-26.2±2.3

52.83±1.3

SA-P-HSA

25

-

HSA

186±42

29.4±3.9

61.37±1.9

SL-P-HSA

-

25

HSA

192±52

-27.8±0.4

66.71±2.4

SA/SL-P-HSA

12.5

12.5

HSA

207±11

16.0±1.7

67.56±5.6

SA-P-F68

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Journal Pre-proof SA: Stearylamine, SL: Soyalecithin, P: PLGA, PVA: Polyvinyl alcohol, HSA: Human serum albumin, TPGS: Tocophenyl polyethylene glycolsuccinate, F-68: Pluoronic 68 Percentage entrapment efficiency The percentage entrapment efficiency (%EE) determines the amount of drug that has entered the carrier and it depends on combinatorial factors. The results of %EE of various formulations are presented in Table 1. The %EE of paclitaxel was found to be higher in the mixture of lipid coated hybrid NPs when compared to their single lipid coated counterparts owing to the longer chain of complex lipid that lead to the increase in size and drug

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entrapment. The %EE of formulations stabilized using PVA and HSA in regard to the single (i.e. SA and SL) and dual lipid (mixture of SA and SL) coating was found not to have any

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significant difference, owing to the potent stabilizer effect demonstrated by PVA and HSA

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[32].

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Fourier transfer infra-red (FTIR) spectroscopy

FTIR analysis was carried out to examine the possible chemical interactions between the drug

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and excipients, if any. The FTIR spectra of the individual excipients and formulations are presented in Fig. 2a and 2b. The results confirmed presence of polymer (PLGA), surfactant

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and the lipid coating on NPs. The C-O stretching peak at 1200 cm-1 confirmed the presence of PLGA in NPs. Lipid coating of synthesized NPs was supported by -CONH- (amide linkage)

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bond formed between the -NH2 groups present on the surface of the NPs (due to the presence of lipid). The characteristic peak at 1730 cm-1 due to the C=O stretching and peak near 1760-1665 cm-1 due to the C=O stretching confirmed the presence of PVA coating and TGPS in synthesized NPs. FTIR spectrum of pure F68 displayed characteristic absorption peaks at 3490 cm−1 and 2890 cm−1 attributed to –OH and C-H bonds. The nanoparticles of F68 with PLGA, SA, SL and SA/SL-P showed characteristics peaks at 1780 cm−1 (C=O), while retaining all other characteristic absorption bands of F68. The amide N-H stretching near 3300-3100 cm-1 revealed the presence of HSA on the surface of the hybrid NPs. All the characteristic peaks of drug were present in the spectrum of formulations indicating no chemical interaction between formulation components.

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Fig. 2a: FTIR spectrum of the formulations (A) Formulations with PVA surfactant (a) Pure PVA (b) P-PVA (c) SA-P-PVA (d) SL-P-PVA and (e) SA/SL-P-PVA (B) Formulations with TGPS surfactant (a) Pure TPGS (b) P- TPGS (c) SA-P-TPGS (d) SL-P-TPGS and (e) SA/SLP-TPGS.

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Fig. 2b: FTIR spectrum of the formulations (C) Formulations with F68 as surfactant (a) Pure F68 (b) P-F68 (c) SA-P-F68 (d) SL-P-F68 and (e) SA/SL-P-F68 (D) Formulations with HSA surfactant (a) Pure HSA (b) P- HSA (c) SA-P-HSA (d) SL-P-HSA and (e) SA/SL-P-HSA.

3.2 Surface morphology To confirm the formation of NPs, TEM images were recorded. TEM images of P-HSA and SA/SL-P-HSA confirmed that the synthesized hybrid NPs are discrete and spherical in shape with smooth surface. TEM observation indicated that the particle size ranged from approximately 150 -185 nm and 206 -225 nm particles, respectively for P-HSA and SA/SL-PHSA NPs (Fig. 3).

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Fig. 3: TEM images of (A) P- HSA and (B) SA/SL-P-HSA

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3.3 In vitro drug release studies

The in vitro release profiles of NPs containing lipid combinations and various surfactants are

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shown in Fig. 4. Initial burst release was observed from all the tested formulations. Lipid

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coated hybrid NPs have shown a controlled release profile when compared to uncoated NPs. The release profile of pure paclitaxel, P-PVA, SA-P-PVA, SL-P-PVA and SA/SL-P-PVA showed 100%, 17%, 13%, 12.7% and 10.4% release within the first 12 h, respectively (Fig.

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4). After 120 h, the formulation P-PVA released 77% drug while the formulation SA-P-PVA, SL-PPVA, SA/SL-P-PVA showed more sustained release i.e. 55%, 48% and 42%,

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respectively owing to the lipid coating. Thus, it could be stated that the drug release from coated NPs was much slower as compared to uncoated ones. The observations of the present study are in accordance with the study by Cheow and Hadinoto. They also recommended mixed lipid coated NPs as the most promising strategy for controlling release rate profile [31]. After 120 h of dissolution study, the drug release profile from the formulations P-F68, SA-PF68, SL-P-F68 and SA/SL-P-F68 was 19%, 13.6%, 10.6% and 10%; from formulations PTPGS, SA-P-TPGS, SL-P-TPGS and SA/SL-P-TPGS was 16.6%, 12%, 14.7%, 11.4%; and from the formulation P-HSA, SA-P-HSA, SL-P-HSA and SA/SL-P-HAS was 21.3%, 18.1%, 15.3%, 14.8%, respectively (Fig. 4). The initial burst release observed was attributed to the dissolution and diffusion of the surface drug [Fig. 4 inserts]. The initial burst could be useful to suppress the development of cancer cells in short time. The result exemplified that the lipid coating of NPs greatly influence the drug release profile. In the present study, the extended

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Journal Pre-proof release profile of paclitaxel was due to the strong hydrophobic interactions of drug with PLGA, which prohibited rapid release of drug from NPs and the encapsulated drug followed a controlled release pattern. Moreover, the drug release from NPs was best fitted with

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Higuchi’s kinetics that describes the diffusion controlled drug release mechanism [33].

Fig. 4: In vitro release profiles of paclitaxel from the NPs containing different surfactants: (A) PVA, (B) HSA, (C) F68, and (D) TGPS in PBS (pH 7.4) containing 5% (w/v) Tween 80 and 20% methanol at 37 ± 0.5 °C. Inserts shows the respective release profiles for a period of 12 h.

3.4 Plasma protein binding study The formation of protein corona results when the plasma proteins get adsorbed on the surface of NPs [7]. In the present study, the extent of protein binding was determined and interpreted with the surface properties of NPs. The percentage protein binding of uncoated NPs i.e. PPVA, P-TPGS, P-F68 and P-HSA were found to be 15.11%, 16.27%, 27.90% and 33.72%, respectively [Fig. 5(A)]. The formulations containing HSA as surfactant showed significantly higher percentage of protein binding when compared to the NPs containing other surfactants. The lipid coating (single and dual) had no influence on protein binding when compared to the 15

Journal Pre-proof uncoated NPs as it remained unchanged. The interaction between the biological environment and lipid NPs can be controlled by surfactant. A coating agent, such as poloxomer, could increase the systemic circulation time of NPs. This can minimize unspecific binding and result in lower uptake of NPs by macrophage. However, it can also decrease the NPs uptake by target cells and result in decreased therapeutic efficacy [34].

Particle size and zeta potential measurements Apart from protein adsorption, physicochemical characteristics such as particle size and zeta potential also influence the cellular uptake of NPs. In the same line, plasma incubated PLGA-

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lipid hybrid NPs were evaluated with respect to particle size and zeta potential (Table 2). The

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zeta potential of protein bound NPs was decreased whereas the particle size increased after the protein binding. The NPs with HSA as surfactant showed higher particle size after the

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plasma exposure, indicating the presence of a large number of plasma protein onto the NPs surface. The decrease in zeta potential of dual lipid coated P-HSA NPs was attributed to the

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increase in percentage protein binding. For instance, Partikel et al reported that PLGA NPs exhibit a zeta potential of -42.3 mV. The zeta potential value of NPs incubated with human

lP

serum and fetal bovine serum decreased to -32.5 mV. A lower concentration of serum was sufficient to form a complete layer of protein [35].

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In vitro release of NPs also varies with the protein binding percentage after the plasma exposure. Higher the protein binding, more sustained release of the drug was observed owing

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to the adsorption of proteins on the surface of NPs. Moreover, the in vitro release profile of P-PVA and SA/SL-P-HSA NPs after co-incubation with plasma showed slower drug release when compared to the release without plasma co-incubation [Fig. 5(B)].

16

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Journal Pre-proof

Fig. 5: (A) Protein binding studies of PLGA NPs (P NPs), stearyl amine coated PLGA NPs

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(SA-P-NPs), soya lecithin coated PLGA NPs (SL-P NPs) and both stearyl amine and soya lecithin coated PLGA-NPs (SA/SL-P-PLGA NPs) using different surfactants; (B) In vitro release profile of paclitaxel from P-PVA NPs, SA/SL-P-PLGA NPs co-incubated with or without plasma in PBS (pH 7.4).

17

Journal Pre-proof Table 2: The effect of protein binding on particle size and zeta potential of NPs. Particle size (nm)

Formulation

Zeta potential (mV)

After

Before

After

P-PVA

355±43

412±25

-38.7±3.4

-3.10±1.5

SA-P-PVA

380±25

440±30

40.2±2.5

1.49±0.9

SL-P-PVA

376±68

435±14

-36.3±1.4

-4.32±1.6

SA/SL-P-PVA

387±34

446±50

24.6±0.6

6.20±2.1

P-TPGS

227±56

293±16

-24.2±3.5

-7.65±2.5

SA-P-TPGS

253±41

305±33

SL-P-TPGS

246±34

311±30

SA/SL-P-TPGS

268±38

325±25

P-F68

261±21

SA-P-F68

285±56

SL-P-F68

272±38

SA/SL-P-F68

298±22

P-HSA SA-P-HSA

SA/SL-P-HSA

2.87±1.2

-27.1±1.3

-5.4±1.5

22.3±0.8

3.18±1.4

331±48

-11.8±2.1

-8.65±2.5

353±32

36.7±1.5

3.77±0.2

342±18

-37.4±1.9

-3.60±0.6

374±27

28.9±0.5

2.51±1.7

168±17

253±29

-26.2±2.3

-1.53±2.4

186±42

276±34

27.4±3.9

2.59±1.3

192±52

290±11

-30.8±0.4

-2.42±2.2

217±11

339±20

16.0±1.7

4.81±1.8

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lP

-p

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36.5±1.9

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SL-P-HSA

of

Before

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code

All value is expressed as mean ± SD; n = 3

3.5 In vitro hemolysis The hemolysis assay is one of the tools that pharmaceutical scientists frequently uses as a guide to represent the toxicity of formulation in blood circulation. Hemolysis is a phenomenon by which the breakdown of RBCs takes place, causing the contained hemoglobin freed into the surrounding medium. The results of hemolysis study of fresh human blood with NPs are summarized in Fig. 6. It was observed that the hemolytic activity of the synthesized NPs depend on the type of surfactant used. The average percentage hemolysis rate of NPs prepared with surfactant PVA, TPGS, F68, HSA were found as 16%, 18

Journal Pre-proof 10%, 13%, 7%, respectively. A decrease in hemolysis rate was observed with an increase in percentage of protein binding. The hemolysis percentage of dual lipid coated HSA NPs (formulation SA/SL-P-PLGA NPs) was below 10%. Similar results for lipid coated nanocomposites on hemolysis percentage have been reported by Han et al [36]. The PVA NPs showed about 2-fold higher hemolysis rates as compared to HSA NPs. In case of proteins adsorbed NPs, the RBCs hemolysis was significantly reduced. This could be due to

lP

re

-p

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the inhibition of interaction of RBCs with the NPs surface.

Fig. 6: Hemolysis activity of PLGA NPs (P NPs), stearyl amine coated PLGA NPs (SA-P-

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NPs), soya lecithin coated PLGA NPs (SL-P NPs) and both stearyl amine and soya lecithin

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coated PLGA-NPs (SA/SL-P-PLGA NPs) by using different surfactant.

3.6 Pharmacokinetic study

The maximum plasma level (Cmax) and time to reach Cmax (tmax) were observed after oral administration of paclitaxel solution and paclitaxel loaded hybrid NPs to the animal models. Plasma

concentration

versus

time

profiles

(described

by

a

non-compartmental

pharmacokinetic analysis) are shown in Figure 7. At the administered dose, Cmax values after oral administration of paclitaxel suspension, P-PVA, P-TPGS, P-F68, P-HSA, SL-P-HSA, SA-P-HSA and SA/SL-P-HSA were 2581, 4247, 3782, 3602, 4241, 7221, 6789 and 7609 ng/ml, respectively. HSA based NPs showed substantial plasma concentration as compared to rest of surfactant based NPs (Fig. 7). The plasma levels of lipid coated NPs were markedly higher than the free paclitaxel solution (Table 3). However, the circulation capability of NPs was different due to surface nature of PLGA NPs. It was observed that the lipid coated NPs had prolonged circulation time than the uncoated NPs. The lipid coated NPs restricted the plasma protein adsorption and minimal complement activation was observed due to the 19

Journal Pre-proof corona coating around the NPs and hence the longer circulation time was achieved. Pandita et al. reported 2-fold and 10-fold higher drug exposure in tissues and plasma, respectively after oral administration of the paclitaxel loaded NPs when compared with the free paclitaxel

lP

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-p

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of

suspension [32].

Fig. 7: Concentration-time profiles in plasma of paclitaxel following oral administration of paclitaxel suspension and paclitaxel loaded NPs at 10 mg/kg to male Wistar rats (n = 3).

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chromatogram of paclitaxel.

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Paclitaxel concentrations in plasma were determined by HPLC assay. Inset shows HPLC

Table 3: Results of pharmacokinetic parameters of paclitaxel after oral administration (10 mg/kg) to male Wistar rats (n = 3). Parameter

Paclitaxel

P-PVA

P-TPGS

P-F68

P-HSA

SL-P-HSA SA-P-HSA SA/SL-P-

suspension

HSA

Tmax (h)

2

6

6

6

6

6

6

6

Cmax (ng/ml)

2581±0.87

4247±0.59

3782±0.75

3602±0.82

4241±1.53

7111±0.72

6789±0.93

7609±0.62

4. Conclusions Paclitaxel loaded polymer-lipid NPs were synthesized by a single step nanoprecipitation approach. The influence of various processing parameters on particle size, surface charge, protein binding and the drug encapsulation efficiency was systematically assessed. The presence of lipids and different surfactants had tremendous influence on the behaviour of 20

Journal Pre-proof NPs. Apart from the increase in %EE and sustained release behaviour, the dual lipid coated hybrid NPs showed higher protein binding, which led to lesser hemolysis rate. The HSA based dual lipid coated NPs showed significantly lower zeta potential and higher protein binding values when compared to the other formulations, and hence, lower percentage of hemolysis. It was observed that the dual lipid coated NPs with HSA as surfactant shows controlled release of paclitaxel with enhanced blood compatibility compared to the NPs with other surfactants. In vivo pharmacokinetic study indicated that lipid coated HSA based NPs showed prolonged circulation time when compared to NPs without a lipid coat. Overall, it can be concluded that the lipid coated NPs can open up a new avenue for the delivery of

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lipophilic bioactives with improved potential.

Acknowledgements

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This work was financially supported by DST, Government of India under the SERB Fast Track Scheme (Grant no. SR/FT/LS-145/2011). The authors would like to thank Fresenius

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Kabi Oncology Ltd., India, for kind support with drug, and Coordinator, DST-FIST, Dept of

Abbreviations

lP

Pharmaceutical Sciences, GJUS & T, Hisar for sample analysis support.

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Nanoparticles (NPs), Poly lactide-co-glycolide (PLGA), Polyvinyl alcohol (PVA), Tocopheryl polyethylene glycol succinate (TPGS), Pluoronic 68 (F68), Human serum

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albumin (HSA) poly(ethylene glycol) (PEG), Ethylene di-amintetraacetic acid (EDTA), dichloromethane (DCM), Stearyl amine (SA), Soya lecithin (SL), Fourier transfer infra-red (FTIR), Potassium bromide (KBr), Percentage entrapment efficiency (%EE), Phosphate buffered saline (PBS), Red blood cells (RBCs), Transmission electron microscopy (TEM).

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Journal Pre-proof Highlights 

Paclitaxel loaded lipid-PLGA hybrid nanoparticles were successfully prepared by single step nanoprecipitation method.



In vitro release behavior of drug from nanoparticles was influenced by lipid coating.



The hemolytic activity of developed nanoparticles was found to be dependent upon the type of surfactant.

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Lipid coated nanoparticles demonstrated prolonged blood circulation.

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

26