Controlled preparation and antitumor efficacy of ...

International Journal of Pharmaceutics 446 (2013) 24–33

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical Nanotechnology

Controlled preparation and antitumor efficacy of vitamin E TPGS-functionalized PLGA nanoparticles for delivery of paclitaxel Guoying Wang a,b , Bo Yu c , Yuequn Wu b , Baolin Huang a,b , Yuan Yuan a,b,∗ , Chang Sheng Liu a,b,∗ a b c

The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China Hangzhou PushiKang Biotechnology Co., Ltd., Zhejiang 310021, PR China

a r t i c l e

i n f o

Article history: Received 12 November 2012 Received in revised form 11 January 2013 Accepted 3 February 2013 Available online xxx Keywords: Paclitaxel TPGS-functionalized PLGA nanoparticles Controllable preparation Drug delivery Antitumor efficacy

a b s t r a c t Vitamin E TPGS-functionalized polymeric nanoparticles have been developed as a promising drug delivery platform in recent years. Obtaining reproducible monodisperse TPGS/polymeric nanoparticles with high encapsulation efficiency (EE%) still remains a big challenge. In this study, an inverse-phase nanoprecipitation method was developed to synthesize TPGS-functionalized PLGA nanoparticles (TPNs) for controlled release of paclitaxel (PTX). To take advantages of lipids, a part of TPGS in the TPNs was replaced by lipids. The results showed that with weight ratio of TPGS-to-PLGA of 2–3 and a molar replacement of lecithin ratio of 30%, the PTX-loaded TPNs (PTPNs) and PTX-loaded lipid-containing TPNs (PLTPNs) exhibited controllable and nearly uniform size of 130–150 nm and EE% of over 80%. Compared to Taxol® , both the PTPNs and PLTPNs significantly increased the intracellular uptake and exerted strong inhibitory effect on human lung cancer A549 model cells. Furthermore, a selective accumulation to tumor site and significant antitumor efficacy of TPNs in the A549 lung cancer xenografted nude mice were observed by intravenous administration, especially for the PTPNs group. Our data suggested that the inverse-phase nanoprecipitation method holds great potential for the fabrication of the paclitaxel-loaded TPNs and the TPNs prepared here is a promising controllable delivery system for paclitaxel. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Paclitaxel (PTX) is one of the best antineoplastic agents and has shown significant clinical activity against a wide variety of tumors like ovarian cancer, lung cancer and breast cancer (Xiao et al., 2012; Takeda et al., 2011). Due to the limited solubility in water, paclitaxel is commercially formulated in the mixture of cremophor EL (Cr-EL) and dehydrated alcohol (1:1, v/v). Unfortunately, this Cr-EL-based formulation often leads to severe side effects including allergic reaction, neurotoxicity, and nephrotoxicity (Yao et al., 2011; Gelderblom et al., 2001). To overcome these disadvantages, various drug delivery systems, such as liposomes (Harmon et al., 2011), nanoparticles (Michael et al., 2008; Garcion et al., 2006), polymer-drug conjugates (Yun et al., 2011) and micelles (Tao et al., 2012) have been developed from different viewpoints. Among them, biodegradable polymeric nanoparticles with coreshell structure within 70–200 nm (Fang et al., 2006; Zhao et al., 2007) due to their high loading capacity for highly insoluble drugs

∗ Corresponding authors at: The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China. Tel.: +86 21 64251358; fax: +86 21 54283420. E-mail addresses: [email protected] (Y. Yuan), [email protected] (C.S. Liu). 0378-5173/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijpharm.2013.02.004

and easily tunable release rate, have attracted much attention for both academic and industrial fields (Zhang et al., 2011a; Song et al., 2011; Hamaguchi et al., 2005). Despite all these appealing features, till now, polymeric nanoparticles have not gained as much success as liposomes in clinic, presumably due to their moderate circulation lifetime and the poor precisely controllable preparation (Zhang and Zhang, 2010). The surface property plays key role on the therapeutic efficacy of nanoparticles. To minimize the adsorption of opsonins and thereafter the removal from the blood, the sufficiently hydrophilic surface is widely applied in nanoparticles (Owen and Peppas, 2006; Salvador-Morales et al., 2009). A series of hydrophilic or amphiphilic polymers have been attempted to decorate the nanoparticle surface, such as poly(ethylene glycol) (PEG) (Chambon et al., 2011), methoxy poly(ethylene glycol) (MPEG) (Zhang et al., 2011b), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Liu et al., 2010) and vitamin E d-a-succinated polyethylene glycol 1000 (vitamin E TPGS) (Akhtar et al., 2011). The particular attention has been given to TPGS, a water-soluble derivative of natural vitamin E, comprising of lipophilic alkyl tail and hydrophilic polar head portion. TPGS not only can act as an excellent emulsifier/absorption enhancer of high emulsification efficiency and cellular adhesion, but also exhibits high hydrophilicity and desirable cellular membrane-penetration capacity (Mu and Feng, 2002,

G. Wang et al. / International Journal of Pharmaceutics 446 (2013) 24–33

2003a,b). In addition, the TPGS itself shows effective toxicity for cancer cells and thus could synergistically enhance the therapeutic efficacy of drug-loaded TPGS-containing nanoparticles (Shieh et al., 2011). However, up to date, the research about the TPGSfunctionalized polymeric nanoparticles was mainly focused on in vitro cellular uptake and cytotoxicity. Little in vivo evaluation has been conducted to show the feasibility and antitumor efficacy of TPNs. To realize effective drug delivery, the particle size and encapsulation efficiency (EE%) of nanocarriers are also important. Generally, the nanoparticles with a size range of 10–150 nm can effectively improve the solubility of poorly water-soluble drugs and prolong the circulation lifetime of drugs. Furthermore, the size range of 100–200 nm is optimal for the enhance permeability and retention (EPR) effect in tumor microenvironment (Perrault et al., 2009). According to the previous reports, most of the polymeric NPs, including TPGS-containing NPs are prepared by the double emulsion or nanoprecipitation process (Farokhzad et al., 2006). The double emulsion process often needs several steps and the production rate is not high. This process often results in a bigger particle size (>200 nm) (Wina and Feng, 2005). As for the traditional nanoprecipitation method, although it can modulate the particle size below 200 nm and EE% near 80%, the process often requires relatively high concentrations of emulsifier (∼5%) and the obtained nanoparticles uniformity is unsatisfactory (Zhang et al., 2008). And the scale-up capability of the traditional nanoprecipitation method is also not desirable. In this study, a modified phase-inverting nanoprecipitation approach was developed to prepare the TPGS-functionalized polymeric nanoparticles (TPNs) with homogeneous particle size in the range of 100–200 nm and high EE% for paclitaxel delivery. PLGA, the most widely used biodegradable polymer was used as the polymer model. In contrast to the frequently-used nanoprecipitation technique, in this phase-inverting nanoprecipitation process, the aqueous phase was added into the organic phase. The resultant PTX-loaded TPGS-functionalized PLGA NPs (PTPNs) showed a very uniform particle size (130–150 nm), and high drug encapsulation (>80%). Taking into account the advantages of lipids in drug delivery, the lipids were also included into our formulation to partially replace TPGS to form a PTX-loaded lipid-TPGS-PLGA system (PLTPNs). The morphology, physical stability, controlled drug release kinetics, in vitro cytotoxicity, in vivo biodistribution and in vivo antitumor efficacy were evaluated for the PTX-loaded nanoparticles.

2. Materials and methods 2.1. Materials TPGS1000 was purchased from Sigma–Aldrich (Shanghai, China). PLGA with a 75:25 monomer ratio, ester-terminated, and viscosity of 0.72 dL/g was purchased from Jinan Dai Gang biological technology Co. (Jinan, China). Paclitaxel was purchased from Knowshine (Shanghai) Pharmachemicals Inc. (Shanghai, China). Taxol® was purchased from Bristol-Myers Squibb (Italy). 6-Coumarin was purchased from Sigma–Aldrich (Shanghai Local Agent, China). DiR was purchased from Caliper Life Sciences (Hopkinton, MA). MTT and phosphate buffer solution (PBS) were purchased from Sigma–Aldrich (Shanghai local agent, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) was received from Gibco (Life Technologies, AG, Basel, Switzerland). A549 cell line was obtained from Shanghai Cell Library of Chinese Academy of Sciences (Shanghai, China). All reagent water used in the laboratory was pretreated with the Milli-Q Plus System (Millipore Corporation, Shanghai Local Agent, China). All solvents including DMSO,

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acetonitrile, acetone and methanol were of HPLC grade and used without further pretreatment. 2.2. Preparation of nanoparticles PTPNs and PLTPNs were synthesized from PLGA, soybean lecithin and TPGS using a modified nanoprecipitation technique. Briefly, PLGA was first dissolved in organic solvent (acetone, unless specified) with concentrations at 4 mg/mL. Pure lecithin and/or TPGS were dissolved in a 4% ethanol aqueous solution in a certain concentration and heated to 65 ◦ C. The preheated aqueous solution was then drop-wise (5 mL/min) added into PLGA acetone solution under gentle stirring with the volume ratio 5:1 and followed by heating at 35 ◦ C for 5 min, then 5 mL distilled water was drop-wised (5 mL/min) into the preheated solution. The nanoparticles were allowed to self-assemble for 20 min with continuous stirring at 35 ◦ C. The remaining organic solvent and free molecules were removed by washing the NP solution 3 times using an Amicon Ultra-4 centrifugal filter (Millipore, Billerica, MA) with a molecular weight cut-off of 10 kDa. The remainder organic solvent was then removed by vacuum rotary evaporation. The NPs were used immediately, stored at −4 ◦ C, or freeze dried in liquid nitrogen and lyophilized for storage at −80 ◦ C for later use. The same procedure was used for the synthesis of fluorescent coumarin-6 loaded TPGSPLGA and TPGS-lecithin-PLGA nanoparticles, except that PTX was replaced by 0.05 wt% coumatin-6. As a comparison, PTPNs were also synthesized by conventional nanoprecipitation process with the identical formulation (Chan et al., 2009). Briefly, PLGA was first dissolved in organic solvent (acetone, unless specified) with concentrations at 4 mg/mL. TPGS were dissolved in aqueous solution in a certain concentration. The PLGA acetone solution was then drop-wise (1 mL/min) added into aqueous solution under gentle stirring with the volume ratio 1:5. The nanoparticles were allowed to self-assemble for 20 min with continuous stirring at 35 ◦ C. The collection and lyophilization of nanoparticles was consistent with the modified nanoprecipitation technique as mentioned in front. 2.3. Characterization of the nanoparticles The size and morphology of paclitaxel-loaded nanoparticles were examined on a JEOL JEM-200CX instrument (TEM) at an acceleration voltage of 200 kV. The sample was prepared by administering the NP suspension (2 mg/mL) onto a 100-mesh formvar-coated copper grid that had been previously hydrophilized under UV light. Samples were stained at room temperature with freshly prepared and sterile-filtered 1% (w/v) phosphotungstic acid aqueous solution. The grids were then air dried prior to imaging. Particle size, polydispersity index, and surface charge were determined by Quasi-elastic laser light scattering using a Zeta PALS dynamic light scattering (DLS) detector (15 mW laser, incident beam 676 nm) (Santa Barbara, CA, USA) at room temperature. Viscosity and refraction indices were set equal to those specific of water. The concentration of paclitaxel was measured with high performance liquid chromatography (HPLC) system with a UV detector (Agilent Technologies Inc, Cotati, CA). An ODS column (Phenomenex, 250 mm × 4.6 mm, 5 ␮m) was used for analysis and the column temperature was kept at 30 ◦ C. The mobile phase was consisted of acetonitrile and water (55/45, v/v). Flow rate was 1.0 mL/min and the detection wavelength was set at 227 nm. All samples were analyzed in triplicate. To estimate the encapsulation efficiency of paclitaxel nanoparticles, the nanoparticles suspensions were destroyed by adding equal volume acetonitrile. The solution was properly diluted prior to HPLC analysis. Encapsulation

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efficiency (EE%) and drug-loading efficient (DL%) were calculated with the following formulas: EE% =

DL% =

Paclitaxel weight measured in NPs × 100% Paclitaxel weight added

Paclitaxel weight measured in NPs × 100% Total weight of NP materials added + Paclitaxel weight added

To study the paclitaxel release rate, PTPNs and PLTPNs and Taxol® injection (containing 500 ␮g paclitaxel) were suspended in 5 mL distilled water and placed in a dialysis tube (12,000–14,000 molecular weight cutoff, Shanghai Yuan Ju biological technology Co, Ltd., Shanghai, China). The dialysis tubes containing nanoparticles, Taxol® were placed into 150 mL PBS at 37 ◦ C, followed by an immediate shaking at a speed of 100 times/min. Phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 8 mM Na2 HPO4 , and 2 mM KH2 PO4 , PBS, pH = 7.4) with 0.5% Tuwen-80 was used as release media. A volume of 1 mL release medium was sampled at appointed time, followed by immediately adding the equal volume of fresh release medium, respectively. The content of paclitaxel was measured by HPLC as above. The release rate (RR) was calculated with the formula: RR = (Wi /Wtotal ) × 100%, where Wi is the measured amount of paclitaxel in release medium at the time-point, and Wtotal is the total paclitaxel amount in the equal volume of NPs suspensions before performing release experiment. 2.4. Cell culture Human lung adenocarcinoma cancer A549 cells (obtained from the Cell Bank of Chinese Academy of Sciences, Shanghai, China) were cultured in 37.5 cm2 flasks with RPMI 1640 medium (Gibco Co., Ltd., Shanghai, China) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Macgene Biotech) at 37 ◦ C with 5% CO2 in a humidified atmosphere. To maintain cells in the exponential growth phase, they were passaged at a ratio of 1:3 every 3 days. Before use, the cells were detached with 0.25% trypsin/0.03% ethylenediamine tetraacetic acid (EDTA) and the cell density was calculated and used at the desired density in later experiments. 2.5. In vitro inhibitory effect on A549 cells The cytotoxicity of the NPs was evaluated using an MTT assay. To measure the inhibitory effects of various paclitaxel formulations on the cancer cells, the A549 cells were seeded into 96-well culture plates at 5000 cells/well and cultured at 37 ◦ C in a humidified atmosphere with 5% CO2 for 24 h. The cells were then exposed to a series of concentrations of Taxol® , PTPNs, PLTPNs. The final concentrations of paclitaxel were in the range of 0–20 ␮g/mL. The blank culture medium was used as a control. The NPs were sterilized with UV irradiation for 60 min before use. After further incubation for 48 h, the cultured cells were assayed for cell viability with MTT. The wells were washed twice with PBS and 30 ␮L of MTT supplemented with culture medium was added. After 3.5–4 h incubation the unreacted dye was removed by aspiration, and 200 ␮L of DMSO was added to each well to dissolve the dark blue crystal. Finally, the absorbance intensity was measured by the microplate reader (Genios, Tecan, Mannedorf, Switzerland) of absorbance wavelength at 570 nm minus background at 660 nm. Cell viability was calculated by the following equation. Cell viability (%) =

Ints × 100% Intcontrol

where Ints is the absorbance intensity of the cells incubated with the NP suspension and Intcontrol is the absorbance intensity of the

cells incubated with the culture medium only (positive control). The Error bars were obtained from triplicate samples. 2.6. Cellular uptake of nanoparticles For a qualitative cellular uptake study, A549 cells were seeded onto chambered coverslips for 24 h. After applying 10.0 mM of free 6-Coumarin, 6-Coumarin nanoparticles (PTPNs, PLTPNs), respectively, cells were further cultured for 2 h. The medium was removed and cells were washed for three times with ice cold PBS. Then, the cells were observed with a laser scanning confocal microscope (Leica SP2, Heidelberg, Germany). 2.7. Antitumor efficacy in lung cancer xenografts Female BALB/c nude mice (initially weighing 16–18 g, were obtained from the Chinese Academy of Sciences Shanghai Animal Experiment Center, shanghai, China) were used for investigating the antitumor efficacy in vivo. Briefly, approximately 1 × 107 A549 lung cancer cells were re-suspended in 200 mL of serumfree medium, and injected subcutaneously into the right flanks of the nude mice. When tumors reached 50–100 mm3 in volume, mice were randomly divided into five treatment groups (eight for each). And physiological saline, Taxol® (10 mg/kg, i.v.), PBS, CON (PTPNs without paclitaxel), PTPNs (10 mg/kg, i.v.), and PLTPNs (10 mg/kg, i.v.) were given to mice, respectively. The mice were then monitored for tumor progression and weight loss every four day. Tumor volumes were calculated as length × width2 /2 (mm3 ). The tumor volume inhibitory rate at day 28 was calculated with the formula: Rv = 1 − (Vdrug /Vsaline ) × 100%, where Vdrug is the tumor volume after treating with drug, and Vsaline is the tumor volume after treated with physiological saline. Changes in body weight of each mouse were also monitored during the treatment to evaluate possible toxic effects of the therapy. 2.8. The tumor accumulation study in tumor-bearing mice In order to observe the tumor accumulation capability of fluorescence DiR labeled PTPNs in mice bearing A549 xenografts, noninvasive optical imaging systems were utilized. Female nude mice (16–18 g, obtained from the Chinese Academy of Sciences Shanghai Animal Experiment Center) were used for in vivo imaging experiment. Subcutaneous tumor models were established by inoculating 1.0 × 107 A549 cells into the right flanks of the nude mice. When tumors reached approximately 500 mm3 in volume, the mice were administered free DiR (2 mg/kg, i.v., diluted in Cremophor EL/ethanol/PBS, 1:1:9, v/v/v), DiR-PTPNs (2 mg/kg, i.v.) and DiR-PLTPNs (2 mg/kg, i.v.). Mice were anaesthetized by intraperitoneal injection of pentobarbital (60 mg/kg), and scanned at 8 h using a Kodak multimodel imaging system (Carestream Health, Inc., USA) with an excitation bandpass filter at 740 nm and an emission at 790 nm. Exposure time was 5 s per image. After in vivo imaging, the mice were sacrificed at 8 h, and the major organs hearts, livers, spleens, lungs, kidneys and tumors were excised. The nearinfrared fluorescence signal intensities in different tissues were measured. 2.9. Statistics analysis Data are presented as the mean ± standard deviation. One-way analysis of variance was used to determine significance among groups, after which post hoc tests with the Bonferroni correction were used for comparison between individual groups. A value of p < 0.05 was considered to be significant.


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Fig. 1. Schematic illustration of the inverse-phase nanoprecipitation method to synthesize TPGS-PLGA hybrid nanoparticles.

3. Results 3.1. Preparation and characterization of the TPNs The modified nanoprecipitation approach proposed for the preparation of TPNs is illustrated in the schematic illustration (Fig. 1). In this process, the aqueous phase containing TPGS was slowly added into the water miscible organic phase. With the slow diffusion of the organic solvent into the aqueous solution, PLGA was precipitated to form nanoparticles and the TPGS coated as the monolayer around the PLGA polymeric core simultaneously. Likewise, PLTPNs was prepared with partial replacement of TPGS by soybean phosphatidylcholine (sPC). The size, polydispersity and morphology of the prepared PTPNs are shown in Fig. 2. It can be seen that the average diameter of the PTPNs obtained was between 120 and 150 nm with a low PDI ∼0.01 (Fig. 2A). The zeta potential was ranged between −15 mV and −20 mV (data not shown), depending on the size and composition of the NPs. Using the conventional nanoprecipitation method with the same formulation, the average diameter of obtained NPs were ranged between 50 and 150 nm with a high PDI ∼0.30 (Fig. 2B). With paclitaxel loading, the particle sizes and zeta potentials still remained in the similar range. TEM images (Fig. 2C and D) revealed that the NPs were dispersed as individual NPs with a well-defined

spherical shape and homogeneous distribution around 100 nm in diameter. Also, the incorporation of PTX did not cause morphological changes. In agreement with the previous report, the particle size observed in the TEM images was smaller than that determined by DLS. This phenomenon may be due to the inherent difference in detection of the particle size between DLS and TEM. The particle size determined by DLS represents their hydrodynamic diameter, whereas that obtained by TEM is related to the collapsed nanoparticles after water evaporation (Yang et al., 2011). 3.2. Optimization of the TPGS/PLGA nanoparticles To achieve optimal PTX encapsulation, the effects of the major preparation conditions on the particle size and EE% were examined. By using 4 mg/mL PLGA in acetone solution and fixing the final aqueous to organic solution volume ratio of 10:1, the mass ratio of TPGS to PLGA polymer was varied from 0.20 to 6, while no lecithin was added. It can be seen from Fig. 3A that the particle size decreased from 170 to 140 nm when the TPGS/PLGA weight ratio was increased from 0 to 2. But at TPGS/PLGA weight ratios greater than 2, the particle size was increased with the increasing of TPGS. In addition, more TPGS in the formulation led to lower PDI, especially when the TPGS/PLGA weight ratio was 2 or higher, the PDI was below 0.1. This result suggests that

Fig. 2. Particles size of the PTPNs prepared by (A) the new nanoprecipitation and (B) the traditional nanoprecipitation. (C and D) Transmission electron microscopy (TEM) images of PTPNs from the new nanoprecipitation with different magnification.

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Fig. 3. Effect of the TPGS/PLGA polymer weight ratio on the nanoparticle size and PDI (A) and the ER (B); effect of the ratio replacement of lecithin on the nanoparticle size and PDI (C) and the ER (D) at a fixed TPGS/PLGA polymer weight ratio 3. The drug EE% was determined by HPLC.

the amount of TPGS was sufficient enough to stabilize the PLGA particles at such weight ratios. Fig. 3B revealed that with the increase of the ratio of TPGS/PLGA, the EE% was correspondingly increased. When the TPGS/PLGA weight ratio was 2 or higher, the EE% was above 80% and the loading of paclitaxel in the NPs was 3% accordingly. Next, at a fixed TPGS/PLGA weight ratio of 3, we further investigated the effect of lecithin replacement on the nanoparticle size and EE%. Along with the increase of the molar ratio of lecithin, the nanoparticle size (Fig. 3C) was reduced and the PDI increased. Meanwhile, as shown in Fig. 3D, with the increase of the substitution degree of the lecithin, the encapsulate rate fell down sharply. Following the increase of lecithin, the rate of TPGS/PLGA was decreased and there are no enough emulsifiers to remain the stability of NPs. Thus, we selected PTPNs at a weight ratio of TPGS-to-PLGA of 3 and a molar replacement of lecithin ratio of 30% for all subsequent nanoparticle preparation unless otherwise noticed.

cryoprotectants such as 10% (wt/vol) sucrose allowed for recovery of 157.0 ± 0.6 nm NPs, similar to the original 144.5 ± 0.5 nm diameters measured by DLS (Fig. 5A). In the absence of sucrose, NPs aggregated significantly to almost 2 ␮m. We continued to perform in vitro assays of long-term stability and protein binding on PTPNs and PLTPNs. Long-term stability were carried out where the NPs were dialyzed in PBS over 120 h at 37 ◦ C. After an initial 5–10 nm increase in size, the NPs maintained size stability throughout the 120 h study. Protein binding were performed in 100% FBS for 120 h at 37 ◦ C. Likewisely, after a slight increase in size in the initial period, the NPs maintained size stability throughout the 120 h study (Fig. 5B).

3.3. Sustained drug release and stability of the nanoparticles The drug loading of paclitaxel was fixed at 3% (w/w) and the in vitro drug release of NPs in PBS was examined for 8 days (Fig. 4). At first glance, paclitaxel released from Taxol® quickly and nearly 100% drug was released after 50 h. But the TPNs without or with lecithin all showed a quick ‘initial burst’ release followed a relative slow release. Approximately 60% of total drug was released in 8 days from NPs, which indicates PTPNs/PLTPNs can provide a longer sustained release time for paclitaxel. In contrast, the release rate of PTX from PTPNs was slightly faster than that from PLTPNs. To investigate the stability of the TPNs prepared, we encapsulated PTX in the NPs (3 wt%), freeze-dried the NP solution in liquid nitrogen and lyophilized the NPs at −80 ◦ C. Addition of

Fig. 4. Comparison of the cell viabilities of A549 cells treated with different paclitaxel containing formulations after 48 h treatment. (vs. Taxol group at same concentration) *p < 0.05.


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different groups. But further increasing the concentration of PTX would lead to significant reduction in A549 cell viabilities on the TPGS-PLGA NPs in comparison with that on the Taxol® at the same concentration. 3.5. In vitro cellular uptake Confocal laser scanning microscopy (CLSM) was employed in this research to visualize the cellular uptake of the coumarin-6 loaded TPNs by the A549 cells after 2 h incubation and the obtained images were presented in Fig. 7. It can be observed that the green fluorescence in (Fig. 7B and C) was obviously brighter than that observed in (Fig. 7A). Since the coumarin-6 contents inside the NPs are the same among all formulations and the pictures were taken under the same exciting laser intensity from the same confocal microscope, the observation indicates that PTPNs/PLTPNs do promote the entry of the NPs into the A549 cells, which is reported a receptor-mediated endocytosis process (Decuzzi and Ferrari, 2007). 3.6. Efficacy in lung cancer xenografts by tail vein injection

Fig. 5. Controlled and sustained release profiles of Taxol® , PTPNs and PLTPNs. Data are presented as the mean–standard deviation (SD).

3.4. In vitro cytotoxicity With Cremophor EL formulation (Taxol® ) as comparison, the viabilities of A549 cells exposed to the same concentration gradient of the drug formulated in vitamin E TPGS-PLGA NPs (PTPNs, PLTPNs) for 48 h were investigated and the results are presented in (Fig. 6). As shown, in lower concentration (0.01–0.5 ␮g/mL), no obvious difference in the inhibitory effects was observed among

In vivo anti-tumor efficacy of TPGS-PLGA NP was evaluated in mice bearing breast xenograft model. The mice were intravenously administered with PBS, TPGS-PLGA NP without PTX, Taxol® , PTPNs and PLTPNs (PTX dose of 10 mg/kg) every 4 days for three consecutive injections with tumors size recorded every other day until the 28th day (Fig. 8). Fig. 8A shows that in A549 xenograft model, PTPNs and PLTPNs achieved superior antitumor effects by tail vein injection compared to Taxol® . In contrast, the PTPNs exhibited the strongest antitumor efficacy in inhibiting the tumor volume (58.8% at day 28). In order to estimate the adverse effects of the PTX formulations, body weight of the mice was also recorded during the treatment and the results are shown in Fig. 8B. It can be easily found that except the Taxol® group, the average body weight of the mice in other groups showed similar strend: slow increase in the initial 8 days, quick increase between 8 and 12 days and afterwards unchanged. But the body weight of sample Taxol® had a significantly reduction in the day 20. At the experimental terminal, the tumor sizes of the PTXtreated groups were all notably smaller than that of the PBS group and TPNs without PTX (Fig. 8C), and followed the same order: PTPNs < PTLPNs < Taxol® < PTPNs without PTX < PBS (Fig. 8D). 3.7. The tumor accumulation study in tumor-bearing mice Fig. 9 represents the tumor accumulation ability of fluorescence DiR-labeled PTPNs in the A549 xenografts bearing mice. After giving DiR-labeled PTPNs as intravenous within 8 h, a strong DiR fluorescence signal was observed in the PTPNs/PLTPNs injected body(Fig. 9A and B), while low fluorescence signal in free DIR injected body (Fig. 9C). Moreover, after giving as intravenous injection at 8 h, major organs (heart, liver, spleen, lung, kidney), and tumor tissues were isolated and observed by the ex vivo images (Fig. 10). Results showed that the strong fluorescence intensity could be found on the tumor tissues after administering PTPNs/PLTPNs by intravenous and followed the same order: PTPNs > PTLPNs > free DiR. These results confirmed that the PTPNs had strong passive tumor target ability through EPR effect. 4. Discussion

Fig. 6. (A) Post formulation stability of the NPs upon liquid nitrogen freeze-drying and lyophilization with 10% sucrose cryoprotectant. (B) In vitro stability of PLGATPGS NPs over 5 days. NPs were incubated with water, 100% PBS and 100% FBS, respectively, at 37 ◦ C. An aliquot of NP suspensions was collected to measure NP size using DLS.

Due to higher emulsification capacity and unique combinatorial therapeutic efficacy, TPGS has been widely used as functionality to decorate NPs (Chen et al., 2011; Liu and Feng, 2011). In this

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Fig. 7. Representative confocal laser scanning microscopy (CLSM) images show the A549 cellular uptake of the fluorescent coumarin-6 loaded nanoparticles (2 h incubation at 10 mM concentration). Left panels represent the phase contrast image and right panels represent the green fluorescence from coumarin-6-loaded nanoparticles in cytoplasm.

study, a inverse-phase nanoprecipitation method was successfully developed to prepare sub-150 nm TPGS-PLGA NPs with high EE (>80%). In the traditional nanoprecipitation process, the water-miscible organic solution containing biodegradable polymer and hydrophobic drug (oil phase) were dropped into aqueous solution (water phase). With the diffusion of organic solvent into aqueous solution, the solid nanoparticles are formed and the organic solvent is removed by evaporation. Due to the low solubility of the polymer in water and the great amount of water used in the system, polymeric droplets are solidified rapidly, often leading to the formation of wider particle size distribution (phase I) (Fig. 11A). In addition, if shear homogenization remains until the division of the large solidified particles into nano-sized fragments without the protection of liquid oil phase, the encapsulated PTX may aspirate into external aqueous phase. As a result, a small particle but low EE% is obtained. As shown in Fig. 2B, the TPNs prepared by the

tradition nanoprecipitation method in our system, showed lager PDI and lower EE% (lower than 80%, data not shown). In this inverse-phase nanoprecipitation process (Fig. 11B), the water phase was dropped into oil phase slowly and the formation of nanoparticles can be divided into two stages. During the initial stage, the volume of organic phase was much larger than the aqueous phase, the formation of nanoparticles was slow and the nanoparticle formed was soft. With the external shear force, the bigger nanoparticles would re-self-assemble into smaller and homogenous nanoparticle. Meanwhile, due to the inherent hydrophobic property, the drug still remained in the particles. In the second stage, with the continuous addition, the volume of water was bigger than that of oil phase, the diffusion of organic phase and thus the solidifying of the pre-solidified droplets would be quickened. Finally, when the volume rate of aqueous phase and organic phase reached 5 times, the nanoparticles would form a stable uniform structure with a low PDI and a minimum amount of drug


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Fig. 8. (A) Antitumor efficacy of NPs in the A549 xenografts in female nude mice and (B) Body weight changes for the tumor-bearing mice after various formulations were given to mice on the indicated days (shown by arrow). Data are presented as the mean ± standard deviation (n > 6). (C and D) Tumor xenografts alignment of each group taken out from the sacrificed A549 mice and the tumor weight at the study end point. Significant differences found between the PTPNs and PLTPNs and the Taxol® groups, and marked as *p < 0.05.

precipitation. It is also found in the experiment that this present method can greatly improve the production rate of the polymeric nanoparticles while not compromising the physicochemical properties of the particles. By this new method, the constructed PTPNs/PLTPNs exhibited the following physicochemical features: small particle size (approximately 140 nm) low PDI (∼0.1) (Fig. 2A), well-defined spherical shape and homogeneous distribution (Fig. 2C and D), high solubility of paclitaxel (>1 mg/mL), and sustained drug release (Fig. 4). This lower release at the initial 2 h (