Surfactant-free biodegradable polymeric nanoparticles generated from self-organized precipitation route: Cellular uptake and cytotoxicity

Accepted Manuscript Surfactant-Free Biodegradable Polymeric Nanoparticles Generated from Selforganized Precipitation Route: Cellular Uptake and Cytotoxicity Ruijing Liang, Liyun Dong, Renhua Deng, Jing Wang, Ke Wang, Martin Sullivan, Shanqin Liu, Jianyin Wang, Jintao Zhu, Juan Tao PII: DOI: Reference:

S0014-3057(14)00173-6 http://dx.doi.org/10.1016/j.eurpolymj.2014.05.017 EPJ 6451

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

12 March 2014 21 May 2014 22 May 2014

Please cite this article as: Liang, R., Dong, L., Deng, R., Wang, J., Wang, K., Sullivan, M., Liu, S., Wang, J., Zhu, J., Tao, J., Surfactant-Free Biodegradable Polymeric Nanoparticles Generated from Self-organized Precipitation Route: Cellular Uptake and Cytotoxicity, European Polymer Journal (2014), doi: http://dx.doi.org/10.1016/ j.eurpolymj.2014.05.017

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Surfactant-Free Biodegradable Polymeric Nanoparticles Generated from Self-organized Precipitation Route: Cellular Uptake and Cytotoxicity Ruijing Liang1, #, Liyun Dong2, #, Renhua Deng1, Jing Wang2, Ke Wang1, Martin Sullivan1, Shanqin Liu1, Jianyin Wang1, Jintao Zhu1, 3,*, and Juan Tao2, *

1

Hubei Key Lab of Materials Chemistry and Service Failure, School of Chemistry and Chemical

Engineering, Huazhong University of Science and Technology (HUST), Wuhan 430074, P. R. China 2

Department of Dermatology, Affiliated Union Hospital, Tongji Medical College, HUST, Wuhan

430022, P. R. China 3

National Engineering Center for Nanomedicine, HUST, Wuhan 430074, P. R. China

* E-mail: [email protected] (J. T. Z.); [email protected] (J. T.) #

These authors contributed equally to this work

1

ABSTRACT: Surfactant-free biodegradable polymeric nanoparticles (NPs) with uniform sizes were prepared by self-organized precipitation (SORP) method. Size and size distribution of the NPs can be easily tuned by varying the preparation conditions. More importantly, we demonstrate that hydrophobic species, semiconductor nanocrystals, and magnetic NPs can be encapsulated into the polymeric NPs effectively and quantitatively to generate multifunctional hybrid NPs. No surfactant is employed during the preparation process, which is crucial for the formed polymeric NPs to be used in bio-related applications. To evaluate the influence of surfactants on cellular behavior, cellular uptake and cytotoxicity of surfactant-free NPs and surfactant-coated NPs were performed. Our results indicated that surfactant-free NPs could be more promptly and effectively phagocytized by cells in vitro compared to residual surfactant-coated NPs prepared from the emulsion-solvent evaporation method, providing a proof that the surfactant-free NPs have more advantages in cellular uptake and more safety in drug delivery and bio-imaging. Moreover, surfactant-coated NPs inhibited cellular uptake of NPs, and had selective toxicity to melanoma A875 cells rather than human umbilical vein endothelial cells (EVC304), especially for surfactant polyoxyethylene octyl phenyl ether (Triton X-100) due to the generation of intracellular reactive oxygen species (ROS). The surfactant-free uniform NPs prepared from SORP, combining desirable characteristics of hydrophobic drugs and functional materials, may prove advantageous in simultaneous drug delivery, imaging and magnetic field manipulation applications. KEYWORDS: Biodegradable Nanoparticles, Uniformity, Multifunctionality, Cytotoxicity, Surfactant Effect

2

1. Introduction Due to their unique biocompatibility and biodegradation [1], biodegradable polymeric nanoparticles (NPs) have broad applications in the fields of drug delivery and release, vaccination, targeting, tumor therapy and diagnostic systems [2-5]. Several methods have been developed to prepare biodegradable NPs, including nanoprecipitation [6], emulsion-solvent evaporation [7], emulsion-diffusion [8], salting out [9], dialysis [10], supercritical fluid technology, and other techniques [11]. Among these methods, nanoprecipitation and dialysis approaches are surfactant-free methods which employed the watermiscible organic solvents (e.g., acetone, THF, DMF, et al.). Generally, both of the methods do not allow for the precise control over size and size distribution of the NPs (polydispersity index, PDI < 0.05) [10, 12]. Yet, size of the polymeric NPs plays an essential role in the properties and final applications of NPs, including biodegradation, drug release, biodistribution, circulation time, clearance and uptake mechanisms, flow properties and cytotoxicity. Meanwhile, particles with narrow size distribution yields higher drug encapsulation efficiency, better control over the dose and release behavior of the encapsulated drugs, better biocompatibility with cells and tissues than polydisperse NPs [13-16]. In order to obtain polymeric NPs with narrow size distribution, selective centrifugation is generally adopted, but the process is usually tedious and inefficient [17]. Thus, it is necessary to develop a facile and effective method to prepare biodegradable polymeric NPs with controllable and uniform size. The emulsion-solvent evaporation method was extensively developed to prepare biodegradable polymeric NPs. Recently, novel emulsification methods have been employed to prepare NPs with narrow size distribution by filtering the initial emulsion droplets [18-21]. Due to the physical generality of the formation mechanism, emulsion-solvent evaporation method is applicable to a wide variety of polymers, e.g., poly-DL-lactic (PDLLA or PLA), polylactic-co-glycolic acid (PLGA), and polycaprolactone (PCL). A further benefit is that hydrophobic species, semiconductor nanocrystals (e.g., quantum dots, QDs), and magnetic NPs (MNPs) can be incorporated separately or simultaneously into the polymeric NPs to generate multifunctional NPs through this approach which can enhance the water solubility of the hydrophobic drug, reduce the heavy metal cytotoxicity, and protect their 3

pharmacodynamic effects [20, 22, 23]. However, surfactants are usually required during the preparation process via an emulsification approach and numerous purification stages are therefore required. Additionally, surfactant is usually difficult to be completely removed in the post-processing, and residual surfactant which is the amount of surfactant physically adsorbed on the surface of NPs or encapsulated in the NPs even after the purification process, can affect drug delivery, biological activities, and even cause cytotoxicity [16, 24-27]. For example, residual poly(vinyl alcohol) (PVA) will influence the pharmaceutical properties of NPs and has relatively lower cellular uptake which could be related to the higher hydrophilicity of the NPs surface [25]. It is found that PLGA NPs coated with PVA through the emulsion-solvent evaporation route displayed lower cellular uptake efficiency, compared to the surfactant-free NPs from the nanoprecipitation method [27]. To increase the biocompatibility and targeting capacity, some strategies like surface modification or functionalization were employed [24, 28, 29], which will certainly increase the complexity of the preparation process and cost. Therefore, a facile and effective route to prepare narrowly distributed surfactant-free biodegradable polymeric NPs is highly desirable. Recently, the field of nanoscience and nanotechnology has developed rapidly, especially in relation to nanocarriers in biological field [2, 30]. Nanotoxicology has now become a critical element in the safety assessment of nanomaterials, particularly nanomedicine [31-34]. Most of the studies that investigated the toxic effect of NPs on the cellular behavior are usually focused on inorganic NPs, such as carbon nanotubes, fullerenes, silica, metal NPs, etc [35, 36]. Yet, the toxicity of biodegradable NPs has still not been extensively investigated due to the well-known biocompatibility and biodegradability. But potential risks might arise from NPs preparation process and formulations [31]. The non-ionic surfactants, such as PVA, polysorbate 80 (Tween 80) and Triton X-100, frequently employed during the preparation process of NPs, have an important influence on the pharmaceutical properties of NPs, such as particle size, zeta potential, PDI, surface hydrophobicity, drug loading efficiency and release behavior [33, 34, 37]. Despite some reports investigating the effect of PVA and Tween 80 on cellular uptake, it is desirable to discuss how the NPs affect the cellular behaviors as opposed to possible 4

cytotoxicity. Thus, it is essential to investigate the effect of surfactant-coated NPs on cellular behavior in a thorough and systematic fashion. Herein, we demonstrate the preparation of uniform biodegradable NPs without surfactant through a self-organized precipitation (SORP) route [38]. This method has recently been used to prepare polymeric NPs, such as polystyrene (PS), poly(methyl methacrylate) (PMMA), polystyrenepolyisoprene (PS-PI), PLA [38, 39], and the polymeric NPs can be spontaneously formed based on nonequilibrium processes during slow evaporation of polymer solution [40]. We tuned the size and size distribution of the particles by changing the preparation conditions, and prepared the multifunctional hybrid NPs by encapsulating the semiconductor nanocrystals and magnetic NPs into the polymeric NPs. Moreover, surfactant-coated NPs with similar surface chemistry and surface charge were prepared via membrane-extrusion emulsification approach [18-20, 41]. We chose three types of non-ionic surfactants (PVA, Tween 80 and Triton X-100) to coat polymeric NPs in order to investigate the effect of surfactants on cellular behaviors in vitro. The melanoma cell line A875 and human umbilical vein endothelial cell line EVC-304 were chosen for investigating cellular uptake and cytotoxicity of the NPs since the NPs are potentially applicable in the therapy of melanoma while the vascular endothelial cells (e.g., EVC-304) would be the first cell which NPs come into contact with when NPs are supposed to be systematically applied to the human body. Further study was also conducted to elucidate the possible mechanism of cytotoxicity of the polymeric NPs. 2. Experimental section 2.1 Materials PLA (or PDLLA, amorphous, Mw = 9.5k, PDI = 2.07; 38k, PDI = 1.42; 91k, PDI = 1.46; 649k, PDI = 2.07; and 1215k, PDI = 1.70) and PLGA (amorphous, molar ratio of 75:25, Mw = 694k, PDI = 2.08) were purchased from Jinan Daigang Biomaterial Co., Ltd, China. PCL (crystalline, Mw: 25k, PDI =1.25) was purchased from Polymer Source Inc., Canada. All the polymers used in this study were ester end capped which will increase the hydrophobic character of the polymers. Cadmium Selenide quantum dots 5

(CdSe QDs) modified with oleic acid were synthesized by the nucleation and growth method [42]. Fe3O4 magnetite NPs (MNPs) modified with oleylamine were synthesized by the thermal decomposition approach [43]. Tween 80 and Triton X-100 were obtained from China National Medicine Co., Ltd. Hepes sodium salt (purity > 99.0%) was purchased from Shanghai Kayon Biol. Tech. Co., Ltd, China. The MTT assay, paraformaldehyde solution and Hoechst 33258 were purchased from China Google Biotech. Co., Ltd. The Annexin V-FITC and PI apoptosis detection kit were obtained from Key GEN Biotech Co., Ltd., China. Nile red (purity > 98%), PVA (Mw = 13k-23k, 87-89% hydrolyzed), and 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) for the detection of oxidative species were purchased from Sigma-Aldrich. 2.2 NPs preparation 2.2.1 Preparation of NPs through SORP: Typically, 0.20 mg PLA was first dissolved in 1 mL of THF (a good solvent for the polymer); then under gentle magnetic stirring at room temperature, 1 mL of water (a precipitant for the polymer) was added slowly to the polymer solution via a syringe pump at a speed of 60 mL/h. The polymer solution remained optically transparent after this mixing process. The resulting polymer solution in a small vial was left open to air at room temperature (25 ºC) until the THF had completely evaporated, and the NPs suspension was thus obtained (shown in Figure 1a). Then, the PLA NPs suspensions were centrifuged at a speed of 1.2×104 rpm for 10 min to remove the supernatant, and the NPs were re-dispersed in deionized water with a total volume of 0.2 mL to keep the concentration of the NPs solution at ~1 mg/mL. Experimental parameters, including initial polymer concentration (CPLA, 0.05-1.0 mg/mL), ratio of water to organic solvent (RW/O, 1:1-3:1), mixing rate (RM, 2-160 mL/h), and polymer molecular weight (Mw, 9.5k-1215k), effect on the size and size distribution of the NPs were systematically investigated. To achieve functional polymeric NPs, oleic acid capped CdSe QDs and oleylamine capped MNPs (0.5 wt % relative to the polymer, respectively) were first dissolved separately or simultaneously with PLA in THF, and a similar preparation procedure was carried out as described above. In addition,

6

hydrophobic Nile red, as a model drug, could also be entrapped in the NPs when 0.2‰ (w/w) Nile red relative to the polymer was added to the PLA/THF solution before the precipitant addition. 2.2.2 Preparation of NPs by emulsion-solvent evaporation route: 10 mg/mL PLA in 100 µL chloroform was first emulsified with 1 mL aqueous solution containing 3 mg/mL of non-ionic surfactant (PVA, Tween 80 or Triton X-100, respectively) by membrane-extrusion emulsification [18-20, 41]. Emulsion droplets with size of ~1-5 µm were then obtained and chloroform was allowed to evaporate by leaving the emulsions in a small beaker open to air at room temperature to obtain the PLA NPs suspension. The resulting PLA NPs suspensions were centrifuged at a speed of 1.2×104 rpm for 10 min to remove the surfactant, and the NPs were re-dispersed in deionized water with a total volume of 1 mL to keep the concentration of the NPs solution at ~1 mg/mL. PLA NPs prepared by SORP were marked as P0 (blank PLA NPs) and P1 (Nile red-loaded PLA NPs). Nile red-loaded PLA NPs prepared from emulsification with PVA, Tween 80 and Triton X-100 were marked as EPVA, ETween 80, and ETriton X-100, respectively. 2.3 NPs characterization Morphology of the resulting polymeric NPs was characterized by scanning electron microscope (SEM, Sirion 200). Mean size, size distribution (e.g., PDI) and zeta potential of the NPs were measured by dynamic light scattering (DLS, Malvern Nano-ZS90). Multifunctional polymeric NPs encapsulated with QDs/MNPs/Nile red were determined by inverted fluorescence microscope (FM, Olympus IX71) and transmission electron microscope (TEM, Tecnai G2-20). Nile red encapsulation efficiency was determined by fluorospectrophotometry (Shimadzu RF-530). For SEM sample preparation, a drop of the dilute polymeric NPs suspension was dropped onto clean silicon wafer, and then coated with a thin layer of platinum after drying. For TEM sample preparation, a drop of the very dilute NPs dispersion was placed onto TEM copper grid covered by a polymer support film pre-coated with a carbon thin film. The sample was allowed to dry in air and at room temperature before observation. To increase the contrast between QDs/MNPs and the polymeric 7

NPs, 1 wt % phosphotungtic acid (PTA) aqueous solution was employed to negatively stain the samples on the TEM copper grid before TEM observation [44, 45]. For NPs size and size distribution determination, a dilute suspension of NPs was dispersed in the deionized water (100 µg/mL) and shaken before the measurement of particle size. To measure zeta potential of NPs, a suspension of NPs was dispersed in a 1 mM Hepes buffer with pH of 7.4, which was adjusted with 0.1 M HCl or 0.1 M NaOH, and the zeta potential of the solution was measured immediately after preparation [25]. 2.4 Residual surfactant determination The amount of PVA associated with NPs after centrifugation as described above was determined by a colorimetric method based on the formation of a colored complex between two adjacent hydroxyl groups of PVA and an iodine molecule. Briefly, 1 mg of lyophilized NPs was treated with 2 mL of 0.5 M NaOH for 15 min at 60 ºC. Each sample was neutralized with 900 µL of 1 M HCl and the volume was adjusted to 5 mL with distilled water. To each sample, 3 mL of a 0.65 M solution of boric acid, 0.5 mL of a mixed solution of I/KI (0.05 M/0.15 M), and 1.5 mL of distilled water were added. Finally, the absorbance of the samples was measured at a fixed wavelength of 690 nm by using a UV 1801 spectraphotometer (Beijing Rayleigh Analytical Instrument Co.) after 15 min incubation. A standard plot of PVA was prepared under identical conditions [25]. The residual amount of Tween 80 was determined by HPLC to test the quantitative hydrolysis of Tween 80 to oleic acid. First, in the hydrolysis process of Tween 80, 0.9 mL NPs suspension (containing 1 mg PLA NPs) was mixed with 0.1 mL of 1 M KOH solution, then this mixture was heated up to 40 ºC with water bath for 6 h. After that, the mixture was cooled down to room temperature and filtered into a HPLC vial. The mobile phase was composed of acetonitrile and buffer solution (60:40, v/v) and the pH of the solution was adjusted to 2.86 ± 0.1 with 85% phosphoric acid. In this case, KOH was employed to hydrolyze Tween 80 to oleic acid while the buffer solution was used to adjust the pH value of the solution. The phosphate solution was prepared by dissolving 2.76 g of potassium phosphate, 8

monobasic in 1 L of purified water. After the preparation process, Tween 80 was determined in triplicate by HPLC (Agilent 1100 series) equipped with a Hypersil ODS2 5 µm column (China Dalian Elite Analytical instrument Co. Ltd.). The column temperature was maintained at 30 ºC. The flow rate was set at 1.0 mL/min and the detection wavelength was 210 nm. Sample solution was injected at a volume of 20 µL with 20 min run time. A standard plot of Tween 80 was prepared under identical conditions [46]. The determination of the residual Triton X-100 (1 mL NPs suspension containing 1 mg PLA NPs as the sample) was carried out on HPLC in triplicate by using acetonitrile/water (70:30, v/v) as mobile phase, flow rate of 1.0 mL/min, detection wavelength of 223 nm, and injection volume of 20 µL with 15 min run time. A standard plot of Triton X-100 was also prepared under identical conditions [47]. To make it concise, we use c1 to represent the residual surfactant concentration of the PLA NPs suspension after being centrifuged once and re-dispersed; C1 represents the residual surfactant percentage content (compared to the initial surfactant input 3 mg/mL) of the PLA NPs suspension after being centrifuged once and re-dispersed. C2 represents the residual surfactant percentage contents of the PLA NPs suspension after being centrifuged twice and re-dispersed and while C2s represents the amount of surfactant in the supernatant from PLA NPs suspension after being centrifuged twice. 2.5 Cell line experiment 2.5.1 Cell culture: Human melanoma cell line (A875 cells, Shanghai Maisha Biotechnology Co. Ltd., China) and human umbilical vein endothelial cell line (EVC-304 cells, China National Cell Bank) were cultured in RPMI1640 medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS, Gibco, Grand Island, NY) and 1% penicillin-streptomycin at 37 ºC at a humidified 5% CO2 and 95% air atmosphere. 2.5.2 Cellular uptake analysis: Qualitative studies. The qualitative study of cellular uptake of PLA NPs with different surface coatings was determined by FM (U-LH 100-3, Olympus). A875 cells were seeded into 6-well blank

9

plates at 5×103 cells/well separately. The cover slips were placed on the bottom of each well before the cells were added. After 24 h incubation, cells were washed twice with PBS (0.1 M, pH 7.4) and the medium without FBS was added. Cells were treated with 100 µg/mL (PLA weight concentration) of P1, EPVA, ETween 80, and ETriton X-100 respectively in growth medium for different time intervals (0 min, 1 min, 10 min, 1 h and 6 h) at 37 ºC. After the appropriate incubation period, cells were washed for 4 times with PBS. Then, cells were fixed with 4% paraformaldehyde solution for 20 min and washed for 3 times with PBS again before staining the nuclei with Hoechst 33258 for 5 min. The cells were subsequently washed for 3 times. Subsequently, the cover slips were sealed and viewed under FM. Quantitative studies. Similar experimental process of A875 cells treated with different Nile redloaded PLA NPs for 6 h was carried out as described above. The relative amount of Nile red-loaded PLA NPs taken up by A875 cells was expressed by the mean fluorescence intensity which was detected using a FACSCalibur flow cytometry (Becton Dickinson, USA). The fluorescence emission was collected in the certain channel, and a gate on forward and side scatter was used to exclude cellular debris. 1×104 cells were detected for each sample and experiments were repeated in triplicate. Data was acquired and analyzed using the CellQuest Pro software (version 4.02, BD Biosciences, CA). 2.5.3 In vitro cytotoxicity: The cytotoxicity of the negative control (normal saline, NS), four kinds of Nile red-loaded NPs (P1, EPVA, ETween 80, and ETriton X-100) and their supernatant was determined with the MTT assay according to the manufacturer’s instructions. The concentration of all the NPs suspension was 100 µg/mL (PLA weight concentration). The supernatant was obtained from the NPs solution after being centrifuged twice at 1.2×104 rpm for 10 min, and its content was determined by a colorimetric method or HPLC as described above. A875 and EVC-304 cells were suspended at 8×103 cells/well in 96-well. After 24 h incubation, cells were washed twice with PBS and fresh culture medium without FBS was added. Afterwards, cells were incubated for 24 h, 48 h and 72 h with the same concentrations of all the samples, then washed twice with PBS to remove remaining NPs and 100 µL of FBS-free culture medium was added. 50 µL of MTT solution was added to each well, and cells were incubated for an additional 4 h at 37 ºC. 100 µL dimethyl sulfoxide was added to each well for formazan dye 10

dissolving. The absorbance of each well was measured at 570 nm by using a micro-plate reader (Tecan Austria GmbH, 5082 Grodig, Austria). 2.5.4 Apoptosis analysis: Apoptosis analysis of cells treated with PLA NPs with different surface coating was performed by an Annexin-V FITC+PI assay. Concentration of all the NPs (without Nile red) solution was 100 µg/mL (PLA weight concentration). A875 cells were seeded in 6-well plates at 1×105 cells/well and incubated for 24 h to settle down. The cells were washed with PBS, fresh medium without FBS was added, and then the cells were incubated with same concentration of all the samples for 24 h, 48 h and 72 h. Then, the cells were collected, washed with PBS, and stained according to the manufacturer’s instructions and analyzed by FACS. 2.5.5 Detection of intracellular reactive oxygen species (ROS): DCFH-DA was used to detect the ROS level in A875 cells treated with the NPs. The concentration of all the NPs (without Nile red) solution was 100 µg/mL (PLA weight concentration). A875 cells were seeded in 6-well plate at 5×104 cells/well and incubated for 24 h. The cells were washed with PBS and treated with the same concentration of all the samples (without Nile red) in a fresh FBS-free medium for 1 h, 4 h, 24 h, 48 h and 72 h. After washing with PBS 3 times, the cells were incubated with 10 µM DCFH-DA for 30 min, washed with fresh FBS-free medium and analyzed by flow cytometry. 2.5.6. Statistical analysis: Statistical analysis was performed using SPSS18.0 (SPSS Inc., Chicago, IL, USA). In the cytotoxicity test, data were expressed as means ± standard deviation and a Student’s t test (two-tailed) was carried out to evaluate the differences of cytotoxicity, cellular uptake and ROS with NPs coated with or without surfactant for A875 cells and EVC-304 cells. Values of p < 0.05 indicated a statistically significant difference. 3. Results and discussion 3.1 Formation of PLA NPs through SORP route Uniform polymeric NPs were generated through a SORP approach. Typically, PLA was dissolved in THF (a good solvent for PLA) to form homogeneous solution (Figure 1a). Precipitant (e.g., deionized 11

water) was added slowly to the above polymer solution, resulting in a transparent solution. In this case, the solution was still colorless and no blue tint was detected, indicating that no polymeric NPs were formed. The polymeric NPs with uniform size were formed during the slow evaporation of the organic solvent afterwards. The DLS results of size distribution of the polymeric NPs and SEM investigation (inset in Figure 1b) indicate that we have successfully produced narrowly distributed PLA NPs with spherical shape and size of 332.07 ± 48.00 nm (PDI of 0.01 ± 0.01, Figure 1b) from the SORP method. The value of zeta potential of NPs can be related to the stability of NPs dispersions. As shown in Table 1, the zeta potential of the NPs from SORP is -54.4 ± 8.0 mV (Table 1), indicating a partial hydrolysis of PLA, leads to the stability of NPs suspension in water. As is well known, PLA is one of the most extensively investigated polymers for drug delivery due to its good biodegradability and biocompatibility in endothelial cells, tissues and organs [1]. It has been reported that NPs (e.g., gold and polystyrene NPs) with positive surface charge will cause hemolysis and blood clotting while particles with negative charges are usually used to prevent the coagulation cascade and blood clotting [48]. It is thus expected that the PLA NPs with negative charge (-54.4 ± 8.0 mV) would be compatible with blood, which will be further investigated in the future in vivo experiment. To form uniform polymeric NPs, two main factors have to be considered: desirable water content and solvent evaporation rate. Also, miscibility of the two solvents is essential for this method, and in order to ensure preferential evaporation of the good solvent, the boiling point of the good solvent must be lower than that of the poor solvent. Based on our results and previous reports [38, 40], a possible mechanism for the formation of uniform polymeric NPs during organic solvent evaporation through this route was proposed, as shown in Figure 1c. As water added gradually, the solubility of the polymer in the mixing solvent decreased and the polymer chain gradually folded compactly and became the small nuclei, known as the nucleation process. The rate at which the small nuclei form is called the nucleation rate, and the number of the small nuclei formed is called the nucleation number. Then, as the good solvent evaporates, the nuclei adsorb the free polymer chains and develop to form the NPs (as called the adsorption and growth process). And the NPs are dispersed in the poor solvent when the good solvent 12

was completely removed, as called the precipitation process. The rate at which the small nuclei develop is known as the growth rate. According to our experimental results, we presume that the nucleation process and the adsorption/growth process are a pair of competitive processes, which significantly affect the formation of the NPs and its size/size distribution. Generally, for the nanoprecipitation process, desolvation and precipitation of polymer molecules occur too quickly to allow the formation of NPs with a narrow size distribution. Compared with the nanoprecipitation method [12, 27], a dynamic equilibrium between dissolved polymer molecules and the formed nuclei of polymer particles exists in the SORP process, which changes constantly altering the composition of the mixed solvent, and thus, may induce the formation of the narrowly distributed NPs (PDI < 0.05). 3.2 Size and size distribution control of NPs by SORP According to the above discussion, the size of the PLA NPs can be easily tuned through the variation of initial polymer concentration (CPLA), ratio of water to THF (RW/O), mixing rate (RM), molecular weight (Mw) and solvent evaporation rate. We have systematically investigated the effect of these experimental parameters on the size of the polymeric NPs. Solvent evaporation rate mainly depends on the RW/O, temperature, vapor pressure, specific surface area, and surface air flow rate. Generally, we perform a slow evaporation rate by putting 2 mL polymer solution in a 5 mL open vial at room temperature (~25 ºC). Firstly, CPLA affects the formation of polymeric NPs significantly. Increasing of CPLA will cause the enlargement in the size of the NPs (Figure 2a). In the case of RW/O = 1:1 and RM = 60 mL/h, CPLA was increased from 0.1 to 1.0 mg/mL. Thus, mean size of the polymeric NPs significantly increased from 231.48 ± 80.39 to 553.3 ± 90.52 nm, which also led to a slight enhancement of size distribution (PDI increased from 0.03 to 0.09, obtained from DLS investigation). In accordance with the mechanism of NPs formation, as CPLA increased, the nucleation rate and the growth rate increased significantly and simultaneously. As the growth process had superiority to nucleation initially, the size of NPs increased obviously. However, as the concentration of PLA increased (up to 0.7 mg/mL), the local PLA

13

concentration became high enough and the nucleation process was more likely to occur; at this point, the rate of nucleation and growth became practically equal, leading to a zero growth rate in size of the NPs. On the other hand, the high concentration of local PLA solution might induce the aggregation and cause a slight enhancement of size distribution of NPs. Secondly, mixing rate (RM) of water to THF plays some role in the size of the NP. Size of the particles distinctly decreased when the RM increased to a certain level, such as 80 mL/h (Figure 2b). The mean size of the NPs decreased from 536.22 ± 62.51 to 118.80 ± 45.46 nm when RM was increased from 4 to 160 mL/h. Presumably, the local aggregation of the polymer chains formed the small nuclei due to high local concentration of water in the mixed solvent. Thus, as RM increased, the nucleation number and the nucleation rate distinctly increased, while the growth rate decreased, leading to a reduction in the size of the particles. On the other hand, there was no significant change in the size of the NPs, keeping in the range of 400 to 450 nm, while altering the ratio of water to THF (RW/O) (Figure 2d). Presumably, a higher RW/O led to lower solvent evaporation rate and might induce the formation of more narrowly distributed NPs, but the nucleation number achieved a degree of saturation and had no significant effect on the NPs size. Thirdly, polymer molecular weight (Mw) has another important effect on the size and size distribution of the NPs (Figure 2c). As Mw increased, size of the NPs decreased correspondingly, from 500 to 300 nm, and polymer with high Mw preferentially precipitated to form small nuclei as it has a lower solubility in the mixed solvent. Thus, the nucleation number and rate played the major role in the size of NPs. When Mw increased continuously, the nucleation number and rate increased rapidly. Yet, when the Mw is larger than 200k, size of the NPs kept nearly constant presumably due to the slight increase of nucleation number and rate. Moreover, polymers with high molecular weight could potentially improve the encapsulation ratio of drugs or QDs as hydrophobic materials can be easily trapped in the long polymer chains during the precipitation process, which indicates that the generality of SORP applied in polymer with different molecular weight might provide a convenient way to generate NPs with varied sizes for drug delivery system. 14

By fully considering and adjusting the above experimental parameters, narrowly distributed NPs with size ranging from ~100 to 600 nm, which is an appropriate range for drug delivery, can be effectively produced through this simple SORP route. 3.3 Generality of SORP approach Due to the physical generality of the formation mechanism, this method is applicable to a wide variety of biodegradable polymers. Similar results can be obtained when PLGA or PCL is used. PLGA NPs with narrow size distribution (mean diameter: 275.3 ± 52.8 nm, PDI: 0.05 ± 0.01, zeta potential: 56.8 ± 7.0 mV, Figure 3a) and PCL NPs with uniform size (mean diameter: 404.7 ± 57.5 nm, PDI: 0.03 ± 0.01, zeta potential: -58.0 ± 6.9 mV, Figure 3b) can also be prepared through this technique, which contributes further possibilities for biodegradable polymers in drug delivery. 3.4 Characteristics of multifunctional NPs from SORP In recent years, CdSe QDs have been extensively used as bio-imaging probes due to their unique optical properties [20]. Encapsulation of the QDs into biodegradable polymeric NPs have proved to significantly reduce the toxicity of QDs [22, 23]. In addition, MNPs encapsulated into PLA particles can retain the magnetic profile of the individual NPs, while resulting in a fast and controllable accumulation under the effect of external magnetic field [8, 20]. During the precipitation process, CdSe QDs and MNPs can be simultaneously encapsulated in PLA NPs due to the hydrophobicity of the ligands on their surfaces. FM and TEM images shown in Figure 4a (insets in the lower left corner and the upper right corner, respectively) indicate that CdSe QDs coated with oleic acid were successfully incorporated in the polymeric NPs (size: 301.0 ± 60.1 nm, PDI: 0.03 ± 0.01). The encapsulated CdSe QDs do not greatly influence in the shape and size of the NPs (as displayed in Figure 4a), which makes it possible to prepare polymeric NPs labeled with semiconductor nanocrystals for drug delivery or bio-imaging. Dark spherical spots within the core of the gray hydrophobic PLA NPs (size: 223.7 ± 51.1 nm, PDI: 0.05 ± 0.01) in TEM image (inset in the upper right corner of Figure 4b) refer to the QDs and MNPs (both have high electron density). QDs and MNPs, which have similar size and shape, are difficult to be 15

distinguished in TEM investigation. Further experiments were performed to confirm the incorporation of both NPs into the PLA NPs. The FM image shown in Figure 4b (inset in the lower left corner) indicates that CdSe QDs were successfully incorporated in the polymeric NPs. Figure 4(c-f) showed the NPs encapsulating MNPs were captured by the magnet field after its placement next to a 1.3 T permanent magnet for 5 min. In comparison, in absence of a permanent magnet, the NPs accumulation re-dispersed immediately (Figure 4f). The combination of QDs and MNPs in a single NP provides a mechanism for researchers to control NPs via magnetic manipulation with long-term optical tracking capability. 3.5 Characteristics of PLA NPs prepared by emulsification In order to investigate the effect of residual surfactant on cellular behavior, three different types of non-ionic surfactants (PVA, Tween 80 and Triton X-100) were employed to prepare PLA NPs with different surfactant coatings (marked as EPVA, ETween

80,

and ETriton

X-100)

by membrane-extrusion

emulsification, as shown in Figure 5(a-c), respectively. Nile red can also be encapsulated in the PLA NPs to prepare the fluorescently-labeled NPs during the emulsification process. The physical characteristics of NPs prepared from different methods were shown in Table 1. Excess surfactant and other impurities were removed from all the samples by centrifugation and the concentration of NPs was kept constant at ~1 mg/mL (PLA weight concentration) before all the physical characterization tests. DLS results of the NPs indicated that the size of the prepared NPs is ~200 nm, which can be phagocytized efficiently in an active cellular internalization way [49] and there was no significant difference in the size of the prepared NPs. Thus, it can be concluded that the cell phagocytosis of the NPs will not be affected by NP size prepared through the above mentioned two routes. Clearly, PDI of the NPs prepared by SORP is much smaller than that from emulsification, which implies that the SORP method is more effective at preparing uniform NPs than emulsification approach. On the other hand, the value of zeta potential of NPs was also measured to investigate the stability of the polymeric NPs and the presence of residual surfactant. As can be seen from Table 1, the zeta

16

potential of the different NPs is about -50 mV (except for EPVA of -13.6 mV), indicating their good stability in Hepes buffer solution. As mentioned above, zeta potential of naked NPs from SORP is -54.4 ± 8.0 mV due to the partial hydrolysis of PLA on their surface. Clearly, coating of nonionic surfactant on the surface of naked PLA NPs will significantly decrease (for polymer surfactant, e.g., PVA) or nearly maintain (for small molecule surfactant, e.g., Tween and Triton X-100) the surface charging of the NPs. To gain an accurate and quantitative measurement of residual surfactant coating on the NPs, the colorimetric method or HPLC were employed to determine the surfactant content directly. The residual surfactant concentration of the PLA NPs suspension (c1) and the residual surfactant percentage content of the PLA NPs suspension (C1) after being centrifuged once are shown in Table 1 and Figure 5d. C1 was ~3.5-5.5% of initial surfactant content, while the surfactant percentage contents (C2) decreased to ~1.2-1.8% after being centrifuged twice and the surfactant content of the supernatant from the PLA NPs suspension after being centrifuged twice (C2s) was as low as ~1.7% of initial content. At such low surfactant level, our study shows the effect of a small amount of residual surfactant coated NPs on cellular behavior, which provides a foundation and reference of the polymeric NPs as nanomedicine in the fields of drug delivery, targeting and bio-imaging. 3.6 Cell line experiment 3.6.1 Cellular uptake analysis: Qualitative studies. FM was used to observe cellular uptake and intracellular distribution of Nile red-loaded NPs in A875 cells. After incubation with Nile red-loaded NPs P1 without surfactant, weak red fluorescence was detected in the cytoplasm of most cells after 1 min (Figure 6). After 10 min incubation, stronger red fluorescence was detected in the cytoplasm of all the cells; when incubated for 1 h, most of the cells were full of red fluorescence in the cytoplasm. After 6 h incubation, all the cells were filled with intense red fluorescence. As shown in the results, surfactant-free Nile red-loaded NPs could be promptly and effectively phagocytized by human melanoma cells in vitro and the intensity of uptake increased with the increase of incubation time.

17

To investigate the cellular uptake efficiency of NPs with surfactant and find out the residual surfactant effect on the cellular uptake efficiency, Nile red-loaded NPs (EPVA, ETween 80, and ETriton X-100) were incubated with A875 cells for 6 h. From the results in Figure 7, we could find that the cellular uptake efficiency was ETween

80

> EPVA > ETriton

X-100

by observing the intracellular red fluorescence

intensity. It was noteworthy that the fluorescence intensity of surfactant-free Nile red-loaded NPs P1 in Figure 6 was much stronger than that of the NPs coated with residual surfactant. These results suggested that residual surfactant could reduce the cellular uptake of NPs and might further influence its biological activity. Quantitative studies. Firstly, Nile red encapsulation efficiency of different kinds of Nile red-loaded NPs was determined by fluorospectrophotometry, shown in Figure 8a. It was found that P1, ETween 80 and ETriton

X-100

had almost the same encapsulation efficiency except the higher Nile red encapsulation

efficiency of EPVA. PVA, a homopolymer and non-ionic surfactant presenting maximum solubility in water and excellent emulsifying ability, which makes it commonly-used surfactant in the preparation of NPs. Presumably, the lower encapsulation efficiency of Nile red in PLA NPs from SORP than the NPs from emulsion-solvent evaporation route can be ascribed to the limited compatibility between PLA and Nile red which may cause them precipitate individually in the SORP process. Non-encapsulated Nile red can dissolve in water and be removed by centrifugation since the concentration of non-encapsulated Nile red in the supernatant (< input of 0.2 mg/L) is lower than the maximum solubility of Nile red in water (318 mg/L). To confirm the above finding of cellular uptake from FM, the cellular uptake efficiency of surfactant-free and surfactant-coated NPs was quantified by the mean fluorescence intensity of A875 cells through a FACSCalibur cell analyzer. After 6 h incubation, the cellular uptake efficiency was P1 ＞ ETween 80 ＞ EPVA ＞ ETriton X-100 (Figure 8b). These quantitative results were consistent with the FM images, which demonstrated that residual surfactant coated on NPs could suppress cellular uptake of NPs and different surfactant coatings had varied influence in cellular uptake efficiency. Considering the 18

Nile red encapsulation efficiency, P1 had a lower Nile red loading but the highest cellular uptake efficiency, indicating the great advantage of surfactant-free NPs from SORP. Previous studies have shown that some surfactant may affect the cellular uptake of NPs. Boury and coworkers demonstrated that PVA binding to the surface of the NPs couldn’t be removed entirely by washing [51]. Sahoo et al. showed that the residual PVA on the PLGA NPs significantly decreased the intracellular uptake of NPs in human arterial smooth muscle cells [25], which was consistent with our results. Yet, Tween 80-coated NPs were found being phagocytized by brain endothelial cells more rapidly and in higher amounts than uncoated NPs [37]. This finding was in contrast with our results and the possible explanation was that different systems, including cells and the NPs, were investigated. More work is needed to investigate the reason for the difference, which is certainly beyond the scope of this work. Up to now, there has been no detailed study on the comparison of cellular uptake efficiency between surfactant-free and non-ionic surfactant-coated NPs (e.g., PVA, Tween 80 and Triton X-100). Our study found that surfactant coating could affect the cellular uptake efficiency of NPs in tumor cells, especially Triton X-100, which sheds some light on the optimization choice of surfactant in preparing NPs for drug delivery system. 3.6.2 In vitro cytotoxicity: The in vitro cytotoxicity was investigated using the MTT assay on A875 and EVC-304 cells treated with NPs with or without surfactant for 24 h, 48 h and 72 h, respectively (Figure 9). For A875 cells, surfactant-free NPs showed no evidence of cytotoxicity from 24 h to 72 h. For these three kinds of surfactant-coated NPs, no cytotoxicity was found in the first 24 h. Yet, after 48 h, they exhibited weak inhibitory effect on cells, and cell viabilities of EPVA, ETween 80 and ETriton X-100 were 90.5%, 95.9% and 81.9%, respectively. After 72 h, the inhibitory effect of surfactant-coated NPs became more remarkable with cell viabilities of 88.9%, 91.5% and 69.8%, respectively, even showing apparent cytotoxicity in the sample ETriton

X-100.

Although the result showed a time-dependent

cytotoxicity effect of the NPs with residual surfactant, the surfactant-free polymeric NPs exhibited no cytotoxicity, which is advantageous in improving the cellular uptake efficiency with remarkable safety.

19

We compared the cytotoxicity of different surfactant-coated NPs against A875 cells, and found that the inhibitory effect on cell viability followed an order of ETriton X-100 ＞ EPVA ＞ ETween 80, which was in contrast to cellular uptake efficiency. This indicated that different surfactant led to different cytotoxic effect of surfactant-coated NPs to cells. When NPs are supposed to be systematically applied to the human body, the vascular endothelial cells would be the first cells which NPs come into contact with. Therefore, we chose human umbilical vein endothelial cell line EVC-304 for further investigation. More interestingly, the result indicated the surfactant-free NPs P1, surfactant-coated samples EPVA and ETween 80 all displayed no cytotoxicity on EVC-304 cells for 24 h to 72 h. Yet, ETriton X-100 exhibited weak inhibitory effect on EVC-304 cells, with cell viabilities of 95.4%, 93.3% and 90.2% at 24 h, 48 h and 72 h, respectively. The selective toxic effect of NPs on tumor cells would benefit for the applications of the NPs to tumor therapy. Our results indicated that the surfactant-free NPs showed no cytotoxicity to A875 cells, while all three kinds of surfactant-coated NPs exhibited cytotoxicity to an extent, of which the ETriton X-100 sample displayed the most toxicity. In the case of EVC-304 cells, only ETriton X-100 had low cytotoxicity. These findings provide us with some information on the selection of NPs coated with different surfactants to avoid unnecessary side effect as much as possible or get benefit from its side effect in tumor therapy. To exclude the effect of residual surfactant in the NP solution, cytotoxicity studies were performed using the supernatant obtained from being centrifuged twice of the NPs solution, which would contain free surfactant, if any was present. It can be seen in Figure 10 that the supernatant of surfactant-free NPs showed no evidence of cytotoxicity on A875 and EVC-304 cells. Meanwhile, only the supernatant of ETriton X-100 showed little inhibitory effect on A875 cell viability at 72 h and the supernatant of EPVA and ETween 80 did not exhibit inhibition on A875 cells. No toxicity from the supernatant of all the samples was observed on EVC-304 cells. The difference in cell viability between A875 cells and EVC-304 cells was consistent with the above results obtained from the NP samples. Our data indicated that the supernatant contained free surfactant and exhibited a certain degree of cytotoxicity on A875 melanoma 20

cells. The cytotoxicity of surfactant-coated NPs was difficult to avoid because the residual surfactant was hard to be completely removed. Therefore, the surfactant-free NPs had more advantage over surfactant-coated NPs. The cytotoxicity of different kinds of surfactant has been discussed in previous investigations [33, 52-29]. For PVA, Menon et al. investigated the cytotoxicity of free PVA to adult human dermal fibroblasts (HDFs) and human aortic smooth muscle cells (HASMCs) at 24 h [33]. They found that PVA showed certain cytotoxicity at a low concentration of 0.62 mg/mL in the case of HDFs, but more than 80% cells survived up to 50 mg/mL for HASMCs, which indicated that the cytotoxicity level varied with different cell lines and the concentration of PVA. Additionally, Davda and coworkers used PLGA, PVA and BSA to formulate NPs, and no evidence of cytotoxicity was observed on human umbilical vein endothelial cells (HUVECs) in the concentration range of 62.5-500 µg/mL at 48 h [52]. Similarly, in our study, EVC-304 cells showed low toxicity response to the surfactant-coated NPs with the concentration of NPs of 100 µg/mL (PLA weight concentration), though the amount of PVA in cell culture solution would be much less than that of Menon’s and Davda’s experiment (below 20 µg/mL). On the other hand, Tween 80 is widely used in biochemical applications and in foods as an emulsifier [53]. However, it was observed that Tween 80 caused apparent cytotoxicity on A549 lung adenocarcinoma cells at the concentration of 1 mg/mL [54]. Furthermore, Ménard et al. demonstrated that IC50 of Tween 80 was ~250 µg/mL on HUVECs at 72 h and in agreement with previous studies [55] and Tween 80 didn’t show cytotoxicity up to 30 µg/mL [56]. In our case, the concentration of Tween 80 in cell culture solution (below 20 µg/mL) was much lower than that, and exhibited less cytotoxicity to A875 melanoma cells. Also, it was reasonable that ETween 80 did not show any cytotoxicity to EVC-304 cells. Triton X-100 has been used in industrial and household products, and it can lead to cytotoxicity due to cell membrane damage [57-59]. Thus, the cytotoxicity of sample ETriton X-100 to A875 and EVC304 cells in our study is understandable.

21

3.6.3 Possible mechanism of cytotoxicity of NPs: Based on the above result and discussion, it is apparent that ETriton X-100 was the most toxic material among all the NPs. Therefore, further study was conducted to elucidate the possible mechanism of cytotoxicity of NPs. To investigate whether the cell viability was affected in apoptosis way, Annexin-V staining was performed. As shown in Figure 11, our results indicated that no remarkable apoptosis was observed in A875 cells exposed to four different PLA NPs for 24 h, 48 h and 72 h. However, it is implied that the cytotoxicity of these surfactant-coated NPs was independent of apoptosis pathway. Thus, it is considered that cytotoxicity of NPs may be induced from another pathway, such as oxidation stress. Recent studies indicated that ROS generation played an important role in the cytotoxicity of NPs [60]. Therefore, the level of intracellular ROS in A875 cells following exposure to each PLA NP sample (without Nile red) was assessed with DCFH-DA staining method, and a special amount of intracellular ROS was quantified as the fluorescence intensity control. As shown in Figure 12, our data showed no evidence of increasing ROS generation for surfactant-free NPs P0 compared with the NS group. The ROS level of cells treated with these three surfactant-coated NP samples did not increase within 24 h compared to the NS group, and the ROS production of all the groups decreased in a time-dependent manner within 24 h. At 48 h, the ROS level of sample ETween

80

and ETriton

X-100

increased notably,

especially for the ETriton X-100 sample. The ROS generation of this sample (ETriton X-100) showed a dramatic increase at the 72 h time-point, whereas the other samples of P0, EPVA and ETween 80 retained a similar level of ROS with the NS group. Our data showed that as the ROS levels in A875 cells increased, a significant decrease in cell viabilities at 48 h and 72 h of sample of ETriton X-100 could be observed. This suggested that the cytotoxicity of ETriton

X-100

sample might be associated with an increase in the

intracellular ROS. 4. Conclusion A facile and effective approach was applied for preparing uniform and size-controllable biodegradable polymeric NPs without surfactant via SORP method. Size and size distribution of the 22

NPs can be easily tuned by varying the experimental parameters. Hydrophobic species, semiconductor nanocrystals or magnetic NPs can be effectively encapsulated in the polymeric NPs to produce the multifunctional polymeric NPs. Cellular uptake studies and cytotoxicity in vitro showed that surfactantfree NPs were more promptly and effectively phagocytized by cells than the surfactant-coated NPs without cytotoxicity. Also, surfactant-coated NPs inhibited cellular uptake of NPs and had selective toxicity to melanoma A875 cells rather than EVC-304 cells, especially for Triton X-100 due to the intracellular ROS increase, which sheds some light on the choice of surfactant and could potentially benefit their applications to tumor therapy. The surfactant-free polymeric NPs with uniform size generated from SORP approach displaying greater advantages in cellular uptake, could find applications in the fields of drug delivery and release, targeting, detection and bio-imaging. Further experiments in vivo are on the way to characterize its biological activity and evaluate the risks on organ distribution, accumulation and clearance/excretion, degradation and metabolism, and immunogenicity in the living organism. Acknowledgements We gratefully acknowledge funding for this work provided by the National Basic Research Program of China (973 Program, 2012CB932500), National Natural Science Foundation of China (81271751) and Excellent Youth Foundation of Hubei Scientific Committee (2012FFA008). We also thank the HUST Analytical and Testing Center for allowing us to use its facilities.

23

FIGURES

Figure 1. (a) Schematic illustration of experimental procedure for preparing the PLA NPs with uniform size by SORP; (b) DLS result of PLA38k NPs with average size of 332.07 ± 48.00 nm produced from SORP route (inset is the representative SEM image of the NPs); (c) Illustration showing the possible formation mechanism of polymeric NPs during the SORP preparation.

24

Figure 2. Plots showing the relationship between the different experimental parameters and the mean size of the PLA NPs prepared by SORP: (a) varied initial PLA concentration in THF (CPLA, 0.1 - 1.0 mg/mL); (b) varied mixing rate (RM, 2 - 160 mL/h); (c) varied polymer molecular weight (Mw: 9.5k 1215k); and (d) varied ratio of water to organic solvent (RW/O, 1:1 - 3:1). The molecular weight of PLA employed in (a, b and d) is 38k. Error bars represent the standard deviation of the data obtained from 3 experiments.

25

Figure 3. SEM images of PLGA694k NPs with diameter of 275.3 ± 52.8 nm (a) and PCL25k NPs with diameter of 404.7 ± 57.5 nm (b) produced from SORP route.

26

Figure 4. SEM images of PLA38k NPs incorporated with 0.5 wt % QDs (PLA NPs size: 301.0 ± 60.1 nm) (a) and encapsulated with both 0.5 wt % QDs and MNPs (PLA NPs size: 223.7 ± 51.1 nm) (b) prepared by SORP; Insets in the upper right corners in (a) and (b) are the representative TEM images; Insets in the lower left corners in (a) and (b) are the corresponding FM images, and the ex/em wavelength for QDs is 520-550 nm (the green fluorescence filters)/580 nm); (c-f) Photographs of the magnetic fluorescence NPs dispersion before (c) and after (d, e) its placement next to a 1.3 T permanent magnet for 5 min. In the magnetic field, the PLA NPs incorporated with magnetic NPs became magnetized, aggregated into one another, and were captured by the magnet; (e) Photograph shows that the magnet in (d) was removed, and the sample vial was turned 90º; (f) After removal of the permanent magnet, accumulated NPs was re-dispersed immediately.

27

Figure 5. SEM images of PLA38k NPs prepared from emulsion-solvent evaporation approach with different surfactants: (a) PVA; (b) Tween 80; and (c) Triton X-100; (d) the surfactant contents in the NPs suspension compared to the initial input: C1 represents the residual surfactant percentage content of the PLA NPs suspension after being centrifuged once and re-dispersed; C2 represents the residual surfactant percentage content of the PLA NPs suspension after being centrifuged twice and re-dispersed and while C2s represents the amount of surfactant in the supernatant from PLA NPs suspension after being centrifuged twice.

28

Figure 6. Bright-field (first column) and FM (second to fourth column) images of A875 cells treated with surfactant-free Nile red-loaded PLA NPs (P1) produced from SORP for 1 min, 10 min, 1 h and 6 h. The red fluorescence signal originated from Nile red inside the NPs while the blue signal indicated the Hoechst stained nuclei of the cells.

29

Figure 7. Bright-field (first column) and FM (second to fourth column) images of A875 cells treated for 6 h with three kinds of surfactant-coated Nile red-loaded PLA NPs (EPVA, ETween

80,

ETriton

X-100)

produced from emulsion-solvent evaporation approach, respectively. The red fluorescence signal originated from Nile red inside the NPs while the blue signal indicated the Hoechst stained nuclei of the cells.

30

Figure 8. (a) Nile red encapsulation efficiency of different kinds of Nile red-loaded NPs (P1, EPVA, ETween

80,

ETriton

X-100)

determined by fluorospectrophotometry. 0.2‰ (w/w) Nile red dissolved in

chloroform was employed as the reference. (b) The mean fluorescence intensity of A875 cells treated for 6 h with different Nile red-loaded PLA NPs which was obtained from FACS analysis. Error bars represent the standard deviation. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

31

Figure 9. In vitro cytotoxicity of PLA NPs prepared from different methods on (a) A875 and (b) EVC304 cells. Cells were treated with the negative control (normal saline, NS) and four kinds of Nile redloaded NPs (P1, EPVA, ETween 80, and ETriton X-100). Cell viability was determined with the MTT assay for 24 h, 48 h and 72 h. Error bars represent the standard deviation. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

32

Figure 10. In vitro cytotoxicity of the supernatant of the NPs suspensions on (a) A875 and (b) EVC-304 cells, obtained from the PLA NPs solution after being centrifuged twice. Cells were treated with the negative control (normal saline, NS) and the supernatant of four kinds of Nile red-loaded NPs solution (P1, EPVA, ETween 80, and ETriton X-100), respectively. Error bars represent the standard deviation. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

33

Figure 11. FACS analysis of A875 cells apoptosis after incubation with four different kinds of NPs (without Nile red) for 24 h, 48 h, and 72h.

34

Figure 12. Intracellular ROS generation of A875 cells incubated with four different kinds of PLA NPs (without Nile red) for 1 h, 4 h, 24 h, 48 h and 72 h. Error bars represent the standard deviation. (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

35

Table 1. Characterizations of different kinds of PLA NPs prepared from SORP and emulsion-solvent evaporation approach. P0 (blank NPs) and P1 (Nile red-loaded NPs) represent the PLA NPs prepared by SORP route, and other three kinds of Nile red-loaded NPs prepared by emulsion-solvent evaporation approach were marked as EPVA, ETween 80, and ETriton X-100.

Particle size (nm)

Zeta Residual Residual b potential surfactant c1 surfactant C1c (mV) (µg/mL) (% of input)

Sample

Surfactant

Nile red

P0

none

none

232.9±39.6

0.03±0.01 ﹣ 54.4±8.0

0

0

P1

none

0.2‰

237.6±41.3

0.05±0.01 ﹣ 54.6±7.8

0

0

EPVA

PVA

0.2‰

226.0±51.1

0.15±0.08 ﹣ 13.6±7.4

104.5±62.5

3.5±2.1

ETween 80

Tween 80

0.2‰

197.2±37.9

0.11±0.11 ﹣ 51.8±5.8

161.3±54.6

5.4±1.8

ETriton X-100 Triton X-100 0.2‰

259.9±73.9

0.57±0.14 ﹣ 54.4±6.5

107.6±66.4

3.6±2.2

a

PDI

Note: a

Concentration of the surfactant in the emulsion systems is 3 mg/mL in the last three cases.

b

c1 represents residual surfactant concentration of the PLA NPs suspension after being centrifuged once and re-dispersed, determined by a colorimetric method or HPLC. c

C1 represents the residual surfactant percentage content (compared to the initial surfactant input 3 mg/mL) of the PLA NPs suspension after being centrifuged once and re-dispersed.

36

References [1] Anderson JM, Shive MS. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 2012;64:72-82. [2] Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science 2004;303:181822. [3] des Rieux A, Fievez V, Garinot M, et al. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J Control Release 2006;116:1-27. [4] Kamaly N, Xiao ZY, Valencia PM, et al. Targeted polymeric therapeutic nanoparticles: design, development and clinical translation. Chem Soc Rev 2012;41:2971-3010. [5] Janib SM, Moses AS, MacKay JA. Imaging and drug delivery using theranostic nanoparticles. Adv Drug Deliv Rev 2010;62:1052-63. [6] Vauthier C, Bouchemal K. Methods for the preparation and manufacture of polymeric nanoparticles. Pharm Res 2009;26:1025-58. [7] Cleland JL. Solvent evaporation processes for the production of controlled release biodegradable microsphere formulations for therapeutics and vaccines. Biotechnol Prog 1998;14:102-7. [8] Yu X, Zhao Z, Nie W, et al. Biodegradable polymer microcapsules fabrication through a template-free approach. Langmuir 2011;27:10265-73. [9] Kawashima Y, Yamamoto H, Takeuchi H, et al. Properties of a peptide containing DLlactide/glycolide copolymer nanospheres prepared by novel emulsion solvent diffusion methods. Eur J Pharm Biopharm 1998;45:41-8. [10] Jeon H-J, Jeong Y-I, Jang M-K, et al. Effect of solvent on the preparation of surfactant-free poly(DL-lactide-co-glycolide) nanoparticles and norfloxacin release characteristics. Int J Pharm 2000;207:99-108. [11] Mayo AS, Ambati BK, Kompella UB. Gene delivery nanoparticles fabricated by supercritical fluid extraction of emulsions. Int J Pharm 2010;387:278-85. [12] Nehilla BJ, Allen PG, Desai TA. Surfactant-free, drug-quantum-dot coloaded poly(lactide-coglycolide) nanoparticles: towards multifunctional nanoparticles. ACS Nano 2008;2:538-44. [13] Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis of polymeric microspheres. Pharm Res 2008;25:1815-21. [14] Siepmann J, Faisant N, Akiki J, et al. Effect of the size of biodegradable microparticles on drug release: experiment and theory. J Control Release 2004;96:123-34.

37

[15] Gaumet M, Gurny R, Delie F. Localization and quantification of biodegradable particles in an intestinal cell model: the influence of particle size. Eur J Pharm Sci 2009;36:465-73. [16] Shakweh M, Besnard M, Nicolas V, et al. Poly (lactide-co-glycolide) particles of different physicochemical properties and their uptake by peyer's patches in mice. Eur J Pharm Biopharm 2005;61:1-13. [17] Gaumet M, Gurny R, Delie F. Fluorescent biodegradable PLGA particles with narrow size distributions: preparation by means of selective centrifugation. Int J Pharm 2007;342:222-30. [18] Deng R, Liu S, Li J, et al. Mesoporous block copolymer nanoparticles with tailored structures by hydrogen-bonding-assisted self-assembly. Adv Mater 2012;24:1889-93. [19] Xu W, Yu X, Liang R, et al. Generation of polymer nanocapsules via a membrane-extrusion emulsification approach. Mater Lett 2012;77:96-9. [20] Liang R, Wang J, Wu X, et al. Multifunctional biodegradable polymer nanoparticles with uniform sizes: generation and in vitro anti-melanoma activity. Nanotechnology 2013;24:455302. [21] Wei Q, Wei W, Lai B, et al. Uniform-sized PLA nanoparticles: preparation by premix membrane emulsification. Int J Pharm 2008;359:294-7. [22] Zhou S, Deng X, Li X. Investigation on a novel core-coated microspheres protein delivery system. J Control Release 2001;75:27-36. [23] Derfus AM, Chan WCW, Bhatia SN. Probing the cytotoxicity of semiconductor quantum dots. Nano Lett 2004;4:11-8. [24] Win KY, Feng SS. Effects of particle size and surface coating on cellular uptake of polymeric nanoparticles for oral delivery of anticancer drugs. Biomaterials 2005;26:2713-22. [25] Sahoo SK, Panyam J, Prabha S, et al. Residual polyvinyl alcohol associated with poly (D,Llactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake. J Control Release 2002;82:105-14. [26] Mura S, Hillaireau H, Nicolas J, et al. Influence of surface charge on the potential toxicity of PLGA nanoparticles towards Calu-3 cells. Int J Nanomedicine 2011;6:2591-605. [27] Xiong S, Zhao X, Heng BC, et al. Cellular uptake of Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles synthesized through solvent emulsion evaporation and nanoprecipitation method. Biotechnol J 2011;6:501-8. [28] Chen D, Ji J, Shen J. Surface tailoring of poly(lactic acid) membrane via surfacefunctionalized microspheres to promote cytocompatibility. Acta Polym Sin 2004;6:826-30. [29] Wang S, Dormidontova EE. Nanoparticle design optimization for enhanced targeting: monte carlo simulations. Biomacromolecules 2010;11, 1785-95.

38

[30] Ferrari M. Cancer nanotechnology: opportunities and challenges. Nat Rev Cancer 2005;5:16171. [31] Rivera Gil P, Oberdörster G, Elder A, et al. Correlating physico-chemical with toxicological properties of nanoparticles: the present and the future. ACS Nano 2010;4:5527-31. [32] Oberdörster G. Safety assessment for nanotechnology and nanomedicine: concepts of nanotoxicology. J Intern Med 2010;267:89-105. [33] Menon JU, Kona S, Wadajkar AS, et al. Effects of surfactants on the properties of PLGA nanoparticles. J Biomed Mater Res A 2012;100:1998-2005. [34] Lewinski N, Colvin V, Drezek R. Cytotoxicity of nanoparticles. Small 2008;4:26-49. [35] Yang H, Liu C, Yang D, et al. Comparative study of cytotoxicity, oxidative stress and genotoxicity induced by four typical nanomaterials: the role of particle size, shape and composition. J Appl Toxicol 2009;29:69-78. [36] Freese C, Gibson MI, Klok HA, et al. Size- and coating-dependent uptake of polymer-coated gold

nanoparticles

in

primary

human

dermal

microvascular

endothelial

cells.

Biomacromolecules 2012;13:1533-43. [37] Ramge P, Unger RE, Oltrogge JB, et al. Polysorbate-80 coating enhances uptake of polybutylcyanoacrylate (PBCA)-nanoparticles by human and bovine primary brain capillary endothelial cells. Eur J Neurosci 2000;12:1931-40. [38] Liang R, Zhu J. Monodisperse PLA/PLGA nanoparticle fabrication through a surfactant-free route. J Control Release 2011;152:e129-31. [39] Yabu H. Self-organized precipitation: an emerging method for preparation of unique polymer particles. Polym J 2013;45:261-8. [40] Yabu H, Higuchi T, Ijiro K, et al. Spontaneous formation of polymer nanoparticles by goodsolvent evaporation as a nonequilibrium process. Chaos 2005;15:047505. [41] Tangirala R, Revanur R, Russell TP, et al. Sizing nanoparticle-covered droplets by extrusion through track-etch membranes. Langmuir 2007;23:965-9. [42] Yu WW, Peng X. Formation of high-quality CdS and other II-VI semiconductor nanocrystals in noncoordinating solvents: tunable reactivity of monomers. Angew Chem Int Ed 2002;41:2368-71. [43] Xu Z, Shen C, Hou Y, et al. Oleylamine as both reducing agent and stabilizer in a facile synthesis of magnetite nanoparticles. Chem Mater 2009;21:1778-80. [44] Zhu J, Ferrer N, Hayward RC. Tuning the assembly of amphiphilic block copolymers through instabilities of solvent/water interfaces in the presence of aqueous surfactants. Soft Matter 2009;5:2471-8. 39

[45] Zhu J, Yu H, Jiang W. Hybridization of poly(4-vinyl pyridine)-b-polystyrene-b-poly(4-vinyl pyridine) aggregates in dioxane/water solution. Eur Polym J 2008;44:2275-83. [46] Hu M, M. Niculescu M, Zhang XM, et al. High-performance liquid chromatographic determination of polysorbate 80 in pharmaceutical suspensions. J Chromatogr A 2003;984:2336. [47] Karlsson G, Hinz AC, Henriksson E, et al. Determination of Triton X-100 in plasma-derived coagulation factor VIII and factor IX products by reversed-phase high-performance liquid chromatography. J Chromatogr A 2002;946:163-8. [48]. DeJong WH, Borm PJA, Drug delivery and nanoparticles: applications and hazards. Int J Nanomed 2008;3:133-49. [49] Shang L, Nienhaus K, Nienhaus GU. Engineered nanoparticles interacting with cells: size matters. J Nanobiotechnol 2014;12:5. [50] He C, Hu Y, Yin L, et al. Effects of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 2010;31:3657-66. [51] Boury F, Ivanova T, Panaïotov I, et al. Dyanmic properties of poly(DL-lactide) and polyvinyl alcohol monolayers at the air/water and dichloromethane/water interfaces. J Colloid Interf Sci 1995;169:380-92. [52] Davda J, Labhasetwar V. Characterization of nanoparticle uptake by endothelial cells. Int J Pharm 2002;233:51-9. [53] Ema M, Hara H, Matsumoto M, et al. Evaluation of developmental neurotoxicity of polysorbate 80 in rats. Reprod Toxicol 2008;25:89-99. [54] Tahara K, Yamamoto H, Kawashima Y. Cellular uptake mechanisms and intracellular distributions of polysorbate 80-modified poly (D,L-lactide-co-glycolide) nanospheres for gene delivery. Eur J Pharm Biopharm 2010;75:218-24. [55] Hirama S, Tatsuishi T, Iwase K, et al. Flow-cytometric analysis on adverse effects of polysorbate 80 in rat thymocytes. Toxicology 2004;19:137-43. [56] Ménard N, Tsapis N, Poirier C, et al. Physicochemical characterization and toxicity evaluation of steroid-based surfactants designed for solubilization of poorly soluble drugs. Eur J Pharm Sci 2011;44:595-601. [57] Berthod A, Tomer S, Dorsey JG. Polyoxyethylene alkyl ether nonionic surfactants: physicochemical properties and use for cholesterol determination in food. Talanta 2001;55:6983.

40

[58] Preté PSC, Gomes K, Malheiros SVP, et al. Solubilization of human erythrocyte membranes by non-ionic surfactants of the polyoxyethylene alkyl ethers series. Biophys Chem 2002;97:4554. [59] Grant RL, Yao C, Gabaldon D, et al. Evaluation of surfactant cytotoxicity potential by primary cultures of ocular tissues: I. Characterization of rabbit corneal epithelial cells and initial injury and delayed toxicity studies. Toxicology 1992;76:153-76. [60] Xia T, Knsnoovochich M, Brant J, et al. Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm. Nano Lett 2006;6:1794-807.

41

Graphical Abstract:

Highlights: •

We apply SORP route to produce uniform surfactant-free biodegradable polymeric NPs.

•

Size and size distribution of NPs can be tuned by varying experiment conditions.

•

Functional species can be encapsulated to produce multifunctional NPs.

•

In vitro cellular uptake and cytotoxicity of different NPs are conducted.

•

Possible mechanism of cytotoxicity of the NPs is also investigated.

42