Article pubs.acs.org/molecularpharmaceutics
Globular Protein-Coated Paclitaxel Nanosuspensions: Interaction Mechanism, Direct Cytosolic Delivery, and Significant Improvement in Pharmacokinetics Yongji Li,†,‡ Zhannan Wu,†,‡ Wei He,*,†,‡ Chao Qin,†,‡ Jing Yao,†,‡ Jianping Zhou,†,‡ and Lifang Yin*,†,‡ †
State Key Laboratory of Natural Medicines and ‡Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China S Supporting Information *
ABSTRACT: About 40% of the marketed drugs and 70−90% of new drug candidates are insoluble in water and therefore poorly bioavailable, which significantly compromises their therapeutic effects. A formulation of nanosuspensions achieved by reducing the pure drug particle size down to seb-micron range is one of the most promising approaches to overcome the insolubility. However, the nanosuspension formulations are subject to instability because of nucleation and particle growth. Therefore, a stabilizer is needed to be incorporated into the nanosuspension formulation during the preparation process to suppress the aggregation of drug particles. β-LG, a globular protein, is broken by heat-induced denaturation, and its hydrophobic area is exposed, which allows it to associate with organic particles. PTX, an insoluble drug, is widely used for the clinical treatment of human cancer. However, this drug’s clinical application is greatly limited by intrinsic defects including poor solubility, adverse side effects, and poor tumor penetration. In this study, we prepared β-LG-stabilized PTX nanosuspensions (PTX-NS) by coating the protein onto nanoscaled drug particles, investigating the stabilization effect of β-LG on PTX-NS, and evaluating its in vitro and in vivo performance. PTXNS with a diameter of approximately 200 nm was easily prepared. β-LG produced significantly stabilized effect on PTX-NS via the interaction between the hydrophobic area of the protein and the hydrophobic surface of the drug particles, which resulted in a conformational change of the protein, the loss of both secondary and tertiary structures, and the transition of Trp residues to a less hydrophobic condition. Importantly, unlike other conventional nanoparticles, PTX-NS could directly translocated across the membrane into the cytosol in an energy-independent manner, without entrapment within the endosomal−lysosomal system. Moreover, compared with Taxol, PTX-NS increased AUC and Cmax by 26- and 16-fold, respectively, and prolonged T1/2 by 314fold. As expected, PTX-NS had better in vitro and in vivo antitumor activity compared to PTX alone. Additionally, β-LG is cytoand bio-compatible, and PTX-NS is not toxic to healthy tissues. In conclusion, the present study has suggested the high potency of globular proteins, such as β-LG, as novel biomaterials for nanosuspension platform to improve the drug delivery for disease treatment. KEYWORDS: globular protein, protein conformation changes, paclitaxel, nanosuspension, pharmacokinetics, cytosolic delivery, tumor therapy
■
INTRODUCTION
including breast, lung, ovarian, and nonsmall cell lung carcinoma. However, similar to many other anticancer drugs, the clinical application of PTX is greatly hampered by its inherent drawbacks such as the poor solubility, adverse side effects, and poor tumor penetration.3,4 To solubilize PTX, a mixture of Cremophor EL and dehydrated ethanol must be formulated (Taxol), and the amount of Cremophor EL is markedly greater for PTX than other drugs containing
Cancer is one of the most devastating diseases worldwide, and it is the leading cause of mortality. Over 10 million new cases will be diagnosed each year, and cancer-related deaths are expected to reach 12 million in 2030.1 Thus, the treatment of cancer represents a great challenge in the twenty-first century. Chemotherapy with anticancer drugs is an important approach for cancer therapy. However, its efficacy is limited by low drug solubility, nonselectivity, toxicity toward healthy organs, reduced efficiency, and drug resistance.2 PTX, an anticancer drug naturally separated from the Taxus brevifolia (western yew) plant, was approved by the FDA in 1992 for the treatment of a broad spectrum of human cancers © XXXX American Chemical Society
Received: December 2, 2014 Revised: March 2, 2015 Accepted: March 23, 2015
A
DOI: 10.1021/mp5008037 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics Cremophor EL.5 Because of the presence of Cremophor EL, this PTX formulation has severe side effects, including allergy, hypersensitivity, and anaphylactic reactions, and these conditions affect 20−40% of patients and compromise the therapeutic index for PTX.6 Cremophor EL also limits tumor penetration of PTX.7 Additionally, special infusion sets, longer infusion time with a large volume of intravenous fluid, and a complicated infusion schedule must be involved in the administration of PTX-formulation containing Cremophor EL.8 Thus, a Cremophor EL-free PTX formulation is highly desirable. Nanosized drug delivery strategies, such as the use of liposomes, polymeric micelles, nanoemulsions, and nanosuspensions (frequently referred to as nanocrystals), have great potential to address the problems associated with drug formulation because they have high loading capacity, enhanced bioavailability and drug stability, less patient variability, long circulation time in the blood, and targeted delivery.9−11 Among the nanosized drug delivery strategies, nanosuspensions obtained by formulating poorly water-soluble drugs into nanoscaled particles represents the most compromising approach due to their high drug loading capacity and ease of preparation;12,13 more than five nanosuspension products have entered the market since 1996. However, the nanosuspension formulation is thermodynamically unstable, with a tendency toward agglomeration or crystal growth. Thus, conventional stabilizers that can produce a steric or electrostatic effect, such as PVP, HPMC, TPGS, and Tweens, must be coated onto the surface of drug particles. Nevertheless, most of these stabilizers do not have a strong stabilization effect, and they cannot be used for intravenous delivery due to toxicity.12,14 In addition to traditional stabilizers, serum proteins, such as albumin, are also used to stabilize nanosuspensions.13,15 Since the albumin-stabilized nanosuspension of PTX (Abraxane) was approved by the FDA in 2005, increasing attention has been paid to the use of plasma proteins for drug delivery because plasma proteins can bind to specific surface receptors that are often overexpressed in tumorous cells, such as the transferrin receptor, the folate receptor, and the epidermal growth factor receptor, thus enhancing the antitumor effects.16,17 Moreover, the decoration of proteins helps decrease the removal of nanoparticles from the blood due to recognition by the reticuloendothelial system or mononuclear phagocytic system,18 which increases the blood circulation half-life of nanoparticles and provides a targeted delivery system.19 To date, only serum proteins are used as stabilizers for nanosuspensions. Few reports indicated that proteins isolated from food could stabilize PTX nanosuspensions. Food proteins, such as soybean protein isolate, whey protein isolate, and β-LG, all of which are globular proteins, are widely used in formulated foods because they have high nutritional value and are generally recognized as safe.20 They have various functional properties including emulsification, gelation, foaming, and water binding capacity. Of these properties, the amphiphilic property is especially interesting. Previous reports indicate that these proteins are promising materials for constructing nanoparticles, including nanoemulsions, core−shell nanoparticles, and nanosuspensions, due to their amphiphilic properties obtained by heat-induced denaturation and the subsequent exposure of the hydrophobic site.21−26 Among these proteins, β-LG, a 162residue globular protein with an isoelectric point pI = 5.2 and a diameter of about 5 nm (Figure 1A), has better flexibility and can absorb rapidly to the hydrophobic interface due to its high
water solubility and unique structure.27 It is stabilized by two disulfide bonds, with nine antiparallel β strands and two αhelixes at the carboxyl terminus (Figure 1B). It has two hydrophobic areas (Figure 1B): one is the hydrophobic pocket of calyx formed by the antiparallel β strands (site I), and the other is near the external hydrophobic pocket located between the α-helix and the β-barrel (site II).28,29 One of the remarkable properties of β-LG is its ability to bind hydrophobic compounds like retinoids and vitamin D to the hydrophobic regions and thus enhance the cellular uptake of bioactive drugs via ligand-binding effects.30−32 Most importantly, our recent findings revealed that the nanocarriers based on β-LG could directly penetrate the cell membrane and enter the cytosol without entrapment within the endosomal−lysosomal system via a lipid raft-like pathway.33 Encouraged by these, we hypothesized that β-LG could serve as an amphiphilic biomaterial and thus be capable of stabilizing PTX nanosuspensions (PTX-NS) by its absorption onto the drug particles via the interaction between the hydrophobic sites of protein and hydrophobic surface of drug particles, leading to a significant improvement in PTX delivery and antitumor effects. Previous reports indicated that β-LG could be used as a carrier for hydrophobic compounds; however, few studies focus on that β-LG can in turn act as a functional material binding onto the organic drug particles, therefore facilitating the drug delivery. First, we have provided a better understanding in elucidating the mechanism and relationship of how process conditions affect the diameter of PTX-NS, and in effect the results would afford guidelines for the preparation of other nanosuspnesion stabilized by β-LG or other globular proteins. Indeed, the present results can also be adapted to know of how other proteins associate with the organic particles. Second, most excitingly, we have demonstrated that, unlike other conventional nanoparticles, PTX-NS with rob-like morphology obtained cellular entry by bypassing the endosomal−lysosomal system. Third, we have proved that PTX-NS had better in vivo antitumor activity in comparison with the marked product, owing to its markedly prolonged circulation time in blood and significant increase in bioavailability. Additionally, the protein coating introduces carboxyl and amine moieties, therefore enabling the drug particles to anchor ligands and achieve targeting drug delivery. Overall, the present study has suggested the high potency of globular proteins, especially β-LG, as stabilizers for nanosuspension formulations to improve the drug delivery for disease treatment.
■
MATERIALS AND METHODS Materials. PTX with more than 99% purity was purchased from Yew Biotechnology Co. Ltd. (Jiangsu, China). Taxol (marked product of PTX) was purchased from Bristol-Myers Squibb (China) Investment Co. Ltd. (Shanghai, China). β-LG (No. L3908, 90% purity), IR 783 probe (90% purity), FITC (98% purity), and MTT (98% purity) were obtained from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). A549, H22, HELF, and L-02 cells were purchased from Nanjin Key GEN Biotech Co., Ltd. (Nanjing, China). Fetal bovine serum, RPMI1640, Dulbecco’s Modified Eagle Medium, and Trypsin (more than 6000 units/mg) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). DAPI, Annexin V-FITC/ PI, and Hematoxylin and Eosin Staining Kits were obtained from the Beyotime Institute of Biotechnology (Haimen, China). The In Situ Cell Death Detection Kit was purchased from F. Hoffmann-La Roche Ltd. (Basel, Switzerland).
B
DOI: 10.1021/mp5008037 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
Da, and the release medium was PBS (pH 7.4). At predetermined intervals, samples were withdrawn from the outside of dialysis bag and replaced with an equal volume of fresh PBS. After filtration through a 0.2 μm membrane filter, the quantity of PTX in the samples was calculated using HPLC. The HPLC system (Shimadazu, Tokyo, Japan) consisted of a LC-10AT pump and a SPD10A UV−vis detector (Shimadazu, Tokyo, Japan). Separation was performed using a Diamonsil C18 column (4.6 mm × 250 mm) at 227 nm. The mobile phase was a mixture of acetonitrile and water (65:35, v/v), and it was pumped at a rate of 1.0 mL/min at 40 °C. The injection volume for analysis was 20 μL. CD Spectroscopy and Fluorescence Measurements. The emission spectrum from 300−500 nm was scanned at an excitation wavelength of 295 nm using a fluorescence spectrometer (SHIMADZU RF-5301PC, Japan). The samples for analysis were native β-LG, denatured β-LG, and the PTXNS coated with denatured β-LG (0.25 mg/mL (pH 7.0)). CD spectra were recorded using a J-810 spectrometer (Tokyo, Japan) equipped with a temperature-controlling unit and a quartz cuvette, and the ellipticity was expressed in millidegrees. The parameters for determination were described as follows: bandwidth, 1 nm; response, 1 s; wavelength range, 250−190 nm; scanning speed, 100 nm/min; cell length, 0.1 cm; temperature, 25 °C; and protein concentration, 0.15 mg/mL (pH 7.0). Cellular Uptake and Location. A549 cells were seeded into 24-well plates at a density of 5 × 104 cells/well and cultured for 24 h at 37 °C. Then, the cells were treated with FITC-labeled PTX-NS for 1 and 4 h at 37 °C, or for 24 h at 37 or 4 °C at a fixed PTX concentration ranging from 100−600 ng/mL in PBS. After being digested from the plates and washed three times with PBS, the cells were observed under a fluorescent microscope (Nikon ECLIPSE Ti−S, Japan), and the florescence intensity was determined using a FCM (BD FACSCalibur, USA). To determine intercellular trafficking, A549 cells were seeded on 30 mm glass bottom cell culture. Then, the cells were incubated with FITC-labeled PTX-NS in fresh RPMI-1640 at a PTX concentration of 50 ng/mL for 2 h. After being washed three times with PBS and fixated in 4% paraformaldehyde, the cells were incubated in medium containing LysoTracker Red and DAPI. The cells were observed using CLSM (LEICA TCS SP5 II, Germany). Internalization Mechanism. A549 cells were seeded on 6well plates at a density of 1 × 105 cells/well and cultured for 24 h at 37 °C. Then, the culture medium was replaced with fresh medium containing endocytic pathway inhibitors including Cyto-D (10 μg/mL), nystatin (10 μM), Cpz (10 μg/mL), nocodazole (20 μM), M-CD (2.5 mM), BFA (20 μM), monensin (200 nM), and NaN3 (10 mM) with DG (50 mM). After being saturated with the inhibitors for 30 min at 37 °C, the cells were incubated with FITC-labeled PTX-NS for 2 h. The fluorescence intensity of the cells was measured using FCM after digested from the plates. In Vitro Cytotoxicity. The in vitro cytotoxicity of β-LG, PTX-NS, and Taxol was assessed in A549, H22, HELF, and L02 cells using MTT method. The cells were seeded into 96-well plates at a density of 1 × 104 cells/well and cultured for 24 h at 37 °C to allow attachment. Various concentrations of β-LG and PTX formulations at fixed drug concentrations (2, 20, 200, and 1000 μg/mL) were added to the wells for 24 h. Subsequently, 20 μL of MTT solution (5 mg/mL in PBS) was added to the
SD rats (200−250 g) and ICR mice (18−22 g) were purchased from the College of Veterinary Medicine Yangzhou University (License No: SCXK (Su) 2012−0004, Yangzhou, China). The animals received care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. Experiments followed a protocol approved by the China Pharmaceutical University Institutional Animal Care and Use Committee. Preparation and Characterization of PTX-NS. PTX-NS was fabricated by an antisolvent-precipitation method, as described in previous reports.21,34 Briefly, 30 mg of β-LG was added to a tube with 30 mL of water, and the mixture was stirred for 2 h at room temperature. After adjusting to pH 7 using 0.1 M HCl or 0.1 M NaOH, the protein solution was heated at 100 °C for 0.5 h. Then, 1 mL of 10 mg/mL PTX solution in acetone was poured into the cooled protein aqueous solution (10 mL, less than 4 °C) under vigorous stirring conditions. Next, the sample was sonicated using an ultrasonic probe (20−25 kHz, Scientz Biotechnology Co., Ltd., Ningbo, China) at 500 w for 10 min. Finally, the PTX-NS was evaporated under reduced pressure with a rotating speed of 50 rpm at 25 °C for 20 min to remove residual acetone. FITClabeled PTX-NS was prepared following the same procedure, except β-LG was labeled by FITC before use. To confirm the coating of β-LG onto PTX-NS, we prepared microscale PTX-NS. The preparation procedure was similar to that of nanoscaled PTX-NS, except sonication was performed at 100 w for 1 min. After dilution, one drop of the microscaled particles was placed on the surface of a glass slide and observed by CLSM (LECIA TCS SP5 II, Heidelberg, Germany). Particle size and PI were measured using a 90Plus Particle Size Analyzer (Brookhaven Instruments, Holtsville, NY) at room temperature according to the DLS principle. The PI was used as a measure for size distribution. Raw data were collected over 5 min at 25 °C and at an angle of 90°. The size was expressed with intensity-weighted Gaussian distribution (with Chi-squared value
Globular Protein-Coated Paclitaxel Nanosuspensions: Interaction Mechanism, Direct Cytosolic Delivery, and Significant Improvement in Pharmacokinetics Yongji Li,†,‡ Zhannan Wu,†,‡ Wei He,*,†,‡ Chao Qin,†,‡ Jing Yao,†,‡ Jianping Zhou,†,‡ and Lifang Yin*,†,‡ †
State Key Laboratory of Natural Medicines and ‡Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China S Supporting Information *
ABSTRACT: About 40% of the marketed drugs and 70−90% of new drug candidates are insoluble in water and therefore poorly bioavailable, which significantly compromises their therapeutic effects. A formulation of nanosuspensions achieved by reducing the pure drug particle size down to seb-micron range is one of the most promising approaches to overcome the insolubility. However, the nanosuspension formulations are subject to instability because of nucleation and particle growth. Therefore, a stabilizer is needed to be incorporated into the nanosuspension formulation during the preparation process to suppress the aggregation of drug particles. β-LG, a globular protein, is broken by heat-induced denaturation, and its hydrophobic area is exposed, which allows it to associate with organic particles. PTX, an insoluble drug, is widely used for the clinical treatment of human cancer. However, this drug’s clinical application is greatly limited by intrinsic defects including poor solubility, adverse side effects, and poor tumor penetration. In this study, we prepared β-LG-stabilized PTX nanosuspensions (PTX-NS) by coating the protein onto nanoscaled drug particles, investigating the stabilization effect of β-LG on PTX-NS, and evaluating its in vitro and in vivo performance. PTXNS with a diameter of approximately 200 nm was easily prepared. β-LG produced significantly stabilized effect on PTX-NS via the interaction between the hydrophobic area of the protein and the hydrophobic surface of the drug particles, which resulted in a conformational change of the protein, the loss of both secondary and tertiary structures, and the transition of Trp residues to a less hydrophobic condition. Importantly, unlike other conventional nanoparticles, PTX-NS could directly translocated across the membrane into the cytosol in an energy-independent manner, without entrapment within the endosomal−lysosomal system. Moreover, compared with Taxol, PTX-NS increased AUC and Cmax by 26- and 16-fold, respectively, and prolonged T1/2 by 314fold. As expected, PTX-NS had better in vitro and in vivo antitumor activity compared to PTX alone. Additionally, β-LG is cytoand bio-compatible, and PTX-NS is not toxic to healthy tissues. In conclusion, the present study has suggested the high potency of globular proteins, such as β-LG, as novel biomaterials for nanosuspension platform to improve the drug delivery for disease treatment. KEYWORDS: globular protein, protein conformation changes, paclitaxel, nanosuspension, pharmacokinetics, cytosolic delivery, tumor therapy
■
INTRODUCTION
including breast, lung, ovarian, and nonsmall cell lung carcinoma. However, similar to many other anticancer drugs, the clinical application of PTX is greatly hampered by its inherent drawbacks such as the poor solubility, adverse side effects, and poor tumor penetration.3,4 To solubilize PTX, a mixture of Cremophor EL and dehydrated ethanol must be formulated (Taxol), and the amount of Cremophor EL is markedly greater for PTX than other drugs containing
Cancer is one of the most devastating diseases worldwide, and it is the leading cause of mortality. Over 10 million new cases will be diagnosed each year, and cancer-related deaths are expected to reach 12 million in 2030.1 Thus, the treatment of cancer represents a great challenge in the twenty-first century. Chemotherapy with anticancer drugs is an important approach for cancer therapy. However, its efficacy is limited by low drug solubility, nonselectivity, toxicity toward healthy organs, reduced efficiency, and drug resistance.2 PTX, an anticancer drug naturally separated from the Taxus brevifolia (western yew) plant, was approved by the FDA in 1992 for the treatment of a broad spectrum of human cancers © XXXX American Chemical Society
Received: December 2, 2014 Revised: March 2, 2015 Accepted: March 23, 2015
A
DOI: 10.1021/mp5008037 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics Cremophor EL.5 Because of the presence of Cremophor EL, this PTX formulation has severe side effects, including allergy, hypersensitivity, and anaphylactic reactions, and these conditions affect 20−40% of patients and compromise the therapeutic index for PTX.6 Cremophor EL also limits tumor penetration of PTX.7 Additionally, special infusion sets, longer infusion time with a large volume of intravenous fluid, and a complicated infusion schedule must be involved in the administration of PTX-formulation containing Cremophor EL.8 Thus, a Cremophor EL-free PTX formulation is highly desirable. Nanosized drug delivery strategies, such as the use of liposomes, polymeric micelles, nanoemulsions, and nanosuspensions (frequently referred to as nanocrystals), have great potential to address the problems associated with drug formulation because they have high loading capacity, enhanced bioavailability and drug stability, less patient variability, long circulation time in the blood, and targeted delivery.9−11 Among the nanosized drug delivery strategies, nanosuspensions obtained by formulating poorly water-soluble drugs into nanoscaled particles represents the most compromising approach due to their high drug loading capacity and ease of preparation;12,13 more than five nanosuspension products have entered the market since 1996. However, the nanosuspension formulation is thermodynamically unstable, with a tendency toward agglomeration or crystal growth. Thus, conventional stabilizers that can produce a steric or electrostatic effect, such as PVP, HPMC, TPGS, and Tweens, must be coated onto the surface of drug particles. Nevertheless, most of these stabilizers do not have a strong stabilization effect, and they cannot be used for intravenous delivery due to toxicity.12,14 In addition to traditional stabilizers, serum proteins, such as albumin, are also used to stabilize nanosuspensions.13,15 Since the albumin-stabilized nanosuspension of PTX (Abraxane) was approved by the FDA in 2005, increasing attention has been paid to the use of plasma proteins for drug delivery because plasma proteins can bind to specific surface receptors that are often overexpressed in tumorous cells, such as the transferrin receptor, the folate receptor, and the epidermal growth factor receptor, thus enhancing the antitumor effects.16,17 Moreover, the decoration of proteins helps decrease the removal of nanoparticles from the blood due to recognition by the reticuloendothelial system or mononuclear phagocytic system,18 which increases the blood circulation half-life of nanoparticles and provides a targeted delivery system.19 To date, only serum proteins are used as stabilizers for nanosuspensions. Few reports indicated that proteins isolated from food could stabilize PTX nanosuspensions. Food proteins, such as soybean protein isolate, whey protein isolate, and β-LG, all of which are globular proteins, are widely used in formulated foods because they have high nutritional value and are generally recognized as safe.20 They have various functional properties including emulsification, gelation, foaming, and water binding capacity. Of these properties, the amphiphilic property is especially interesting. Previous reports indicate that these proteins are promising materials for constructing nanoparticles, including nanoemulsions, core−shell nanoparticles, and nanosuspensions, due to their amphiphilic properties obtained by heat-induced denaturation and the subsequent exposure of the hydrophobic site.21−26 Among these proteins, β-LG, a 162residue globular protein with an isoelectric point pI = 5.2 and a diameter of about 5 nm (Figure 1A), has better flexibility and can absorb rapidly to the hydrophobic interface due to its high
water solubility and unique structure.27 It is stabilized by two disulfide bonds, with nine antiparallel β strands and two αhelixes at the carboxyl terminus (Figure 1B). It has two hydrophobic areas (Figure 1B): one is the hydrophobic pocket of calyx formed by the antiparallel β strands (site I), and the other is near the external hydrophobic pocket located between the α-helix and the β-barrel (site II).28,29 One of the remarkable properties of β-LG is its ability to bind hydrophobic compounds like retinoids and vitamin D to the hydrophobic regions and thus enhance the cellular uptake of bioactive drugs via ligand-binding effects.30−32 Most importantly, our recent findings revealed that the nanocarriers based on β-LG could directly penetrate the cell membrane and enter the cytosol without entrapment within the endosomal−lysosomal system via a lipid raft-like pathway.33 Encouraged by these, we hypothesized that β-LG could serve as an amphiphilic biomaterial and thus be capable of stabilizing PTX nanosuspensions (PTX-NS) by its absorption onto the drug particles via the interaction between the hydrophobic sites of protein and hydrophobic surface of drug particles, leading to a significant improvement in PTX delivery and antitumor effects. Previous reports indicated that β-LG could be used as a carrier for hydrophobic compounds; however, few studies focus on that β-LG can in turn act as a functional material binding onto the organic drug particles, therefore facilitating the drug delivery. First, we have provided a better understanding in elucidating the mechanism and relationship of how process conditions affect the diameter of PTX-NS, and in effect the results would afford guidelines for the preparation of other nanosuspnesion stabilized by β-LG or other globular proteins. Indeed, the present results can also be adapted to know of how other proteins associate with the organic particles. Second, most excitingly, we have demonstrated that, unlike other conventional nanoparticles, PTX-NS with rob-like morphology obtained cellular entry by bypassing the endosomal−lysosomal system. Third, we have proved that PTX-NS had better in vivo antitumor activity in comparison with the marked product, owing to its markedly prolonged circulation time in blood and significant increase in bioavailability. Additionally, the protein coating introduces carboxyl and amine moieties, therefore enabling the drug particles to anchor ligands and achieve targeting drug delivery. Overall, the present study has suggested the high potency of globular proteins, especially β-LG, as stabilizers for nanosuspension formulations to improve the drug delivery for disease treatment.
■
MATERIALS AND METHODS Materials. PTX with more than 99% purity was purchased from Yew Biotechnology Co. Ltd. (Jiangsu, China). Taxol (marked product of PTX) was purchased from Bristol-Myers Squibb (China) Investment Co. Ltd. (Shanghai, China). β-LG (No. L3908, 90% purity), IR 783 probe (90% purity), FITC (98% purity), and MTT (98% purity) were obtained from Sigma-Aldrich Co. Ltd. (St. Louis, MO, USA). A549, H22, HELF, and L-02 cells were purchased from Nanjin Key GEN Biotech Co., Ltd. (Nanjing, China). Fetal bovine serum, RPMI1640, Dulbecco’s Modified Eagle Medium, and Trypsin (more than 6000 units/mg) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). DAPI, Annexin V-FITC/ PI, and Hematoxylin and Eosin Staining Kits were obtained from the Beyotime Institute of Biotechnology (Haimen, China). The In Situ Cell Death Detection Kit was purchased from F. Hoffmann-La Roche Ltd. (Basel, Switzerland).
B
DOI: 10.1021/mp5008037 Mol. Pharmaceutics XXXX, XXX, XXX−XXX
Article
Molecular Pharmaceutics
Da, and the release medium was PBS (pH 7.4). At predetermined intervals, samples were withdrawn from the outside of dialysis bag and replaced with an equal volume of fresh PBS. After filtration through a 0.2 μm membrane filter, the quantity of PTX in the samples was calculated using HPLC. The HPLC system (Shimadazu, Tokyo, Japan) consisted of a LC-10AT pump and a SPD10A UV−vis detector (Shimadazu, Tokyo, Japan). Separation was performed using a Diamonsil C18 column (4.6 mm × 250 mm) at 227 nm. The mobile phase was a mixture of acetonitrile and water (65:35, v/v), and it was pumped at a rate of 1.0 mL/min at 40 °C. The injection volume for analysis was 20 μL. CD Spectroscopy and Fluorescence Measurements. The emission spectrum from 300−500 nm was scanned at an excitation wavelength of 295 nm using a fluorescence spectrometer (SHIMADZU RF-5301PC, Japan). The samples for analysis were native β-LG, denatured β-LG, and the PTXNS coated with denatured β-LG (0.25 mg/mL (pH 7.0)). CD spectra were recorded using a J-810 spectrometer (Tokyo, Japan) equipped with a temperature-controlling unit and a quartz cuvette, and the ellipticity was expressed in millidegrees. The parameters for determination were described as follows: bandwidth, 1 nm; response, 1 s; wavelength range, 250−190 nm; scanning speed, 100 nm/min; cell length, 0.1 cm; temperature, 25 °C; and protein concentration, 0.15 mg/mL (pH 7.0). Cellular Uptake and Location. A549 cells were seeded into 24-well plates at a density of 5 × 104 cells/well and cultured for 24 h at 37 °C. Then, the cells were treated with FITC-labeled PTX-NS for 1 and 4 h at 37 °C, or for 24 h at 37 or 4 °C at a fixed PTX concentration ranging from 100−600 ng/mL in PBS. After being digested from the plates and washed three times with PBS, the cells were observed under a fluorescent microscope (Nikon ECLIPSE Ti−S, Japan), and the florescence intensity was determined using a FCM (BD FACSCalibur, USA). To determine intercellular trafficking, A549 cells were seeded on 30 mm glass bottom cell culture. Then, the cells were incubated with FITC-labeled PTX-NS in fresh RPMI-1640 at a PTX concentration of 50 ng/mL for 2 h. After being washed three times with PBS and fixated in 4% paraformaldehyde, the cells were incubated in medium containing LysoTracker Red and DAPI. The cells were observed using CLSM (LEICA TCS SP5 II, Germany). Internalization Mechanism. A549 cells were seeded on 6well plates at a density of 1 × 105 cells/well and cultured for 24 h at 37 °C. Then, the culture medium was replaced with fresh medium containing endocytic pathway inhibitors including Cyto-D (10 μg/mL), nystatin (10 μM), Cpz (10 μg/mL), nocodazole (20 μM), M-CD (2.5 mM), BFA (20 μM), monensin (200 nM), and NaN3 (10 mM) with DG (50 mM). After being saturated with the inhibitors for 30 min at 37 °C, the cells were incubated with FITC-labeled PTX-NS for 2 h. The fluorescence intensity of the cells was measured using FCM after digested from the plates. In Vitro Cytotoxicity. The in vitro cytotoxicity of β-LG, PTX-NS, and Taxol was assessed in A549, H22, HELF, and L02 cells using MTT method. The cells were seeded into 96-well plates at a density of 1 × 104 cells/well and cultured for 24 h at 37 °C to allow attachment. Various concentrations of β-LG and PTX formulations at fixed drug concentrations (2, 20, 200, and 1000 μg/mL) were added to the wells for 24 h. Subsequently, 20 μL of MTT solution (5 mg/mL in PBS) was added to the
SD rats (200−250 g) and ICR mice (18−22 g) were purchased from the College of Veterinary Medicine Yangzhou University (License No: SCXK (Su) 2012−0004, Yangzhou, China). The animals received care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals. Experiments followed a protocol approved by the China Pharmaceutical University Institutional Animal Care and Use Committee. Preparation and Characterization of PTX-NS. PTX-NS was fabricated by an antisolvent-precipitation method, as described in previous reports.21,34 Briefly, 30 mg of β-LG was added to a tube with 30 mL of water, and the mixture was stirred for 2 h at room temperature. After adjusting to pH 7 using 0.1 M HCl or 0.1 M NaOH, the protein solution was heated at 100 °C for 0.5 h. Then, 1 mL of 10 mg/mL PTX solution in acetone was poured into the cooled protein aqueous solution (10 mL, less than 4 °C) under vigorous stirring conditions. Next, the sample was sonicated using an ultrasonic probe (20−25 kHz, Scientz Biotechnology Co., Ltd., Ningbo, China) at 500 w for 10 min. Finally, the PTX-NS was evaporated under reduced pressure with a rotating speed of 50 rpm at 25 °C for 20 min to remove residual acetone. FITClabeled PTX-NS was prepared following the same procedure, except β-LG was labeled by FITC before use. To confirm the coating of β-LG onto PTX-NS, we prepared microscale PTX-NS. The preparation procedure was similar to that of nanoscaled PTX-NS, except sonication was performed at 100 w for 1 min. After dilution, one drop of the microscaled particles was placed on the surface of a glass slide and observed by CLSM (LECIA TCS SP5 II, Heidelberg, Germany). Particle size and PI were measured using a 90Plus Particle Size Analyzer (Brookhaven Instruments, Holtsville, NY) at room temperature according to the DLS principle. The PI was used as a measure for size distribution. Raw data were collected over 5 min at 25 °C and at an angle of 90°. The size was expressed with intensity-weighted Gaussian distribution (with Chi-squared value
