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Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Preparation and characterization of zwitterionic phospholipid polymercoated poly(lactic acid) nanoparticles a
a
ab
c
Li-Li Bao , Hao-Qiang Huang , Jing Zhao , Kenichi Nakashima & ad
Yong-Kuan Gong a
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, Shaanxi, P.R. China b
School of Environmental and Chemical Engineering, Xi’an Polytechnic University, Xi’an 710048, Shaanxi, P.R. China c
Faculty of Science and Engineering, Department of Chemistry, Saga University, 1 Honjo-machi, Saga 840-8502, Japan d
Engineering Laboratory for Bionic Biomaterials and Devices of Xi’an City, Northwest University, Xi’an 710069, Shaanxi, P.R. China Published online: 03 Sep 2014.
To cite this article: Li-Li Bao, Hao-Qiang Huang, Jing Zhao, Kenichi Nakashima & Yong-Kuan Gong (2014) Preparation and characterization of zwitterionic phospholipid polymer-coated poly(lactic acid) nanoparticles, Journal of Biomaterials Science, Polymer Edition, 25:14-15, 1703-1716, DOI: 10.1080/09205063.2014.952993 To link to this article: http://dx.doi.org/10.1080/09205063.2014.952993
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, Nos. 14–15, 1703–1716, http://dx.doi.org/10.1080/09205063.2014.952993
Preparation and characterization of zwitterionic phospholipid polymer-coated poly(lactic acid) nanoparticles
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Li-Li Baoa, Hao-Qiang Huanga, Jing Zhaoa,b, Kenichi Nakashimac and Yong-Kuan Gonga,d* a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, Shaanxi, P.R. China; bSchool of Environmental and Chemical Engineering, Xi’an Polytechnic University, Xi’an 710048, Shaanxi, P.R. China; cFaculty of Science and Engineering, Department of Chemistry, Saga University, 1 Honjo-machi, Saga 840-8502, Japan; dEngineering Laboratory for Bionic Biomaterials and Devices of Xi’an City, Northwest University, Xi’an 710069, Shaanxi, P.R. China
(Received 30 April 2014; accepted 6 August 2014) Poly(lactic acid) (PLA) nanoparticles (NPs) are the most promising polymer NPs for drug delivery and targeting. However, they are easily recognized as a foreign body and rapidly cleared from the body by the mononuclear phagocyte system. Cell membrane mimetic random copolymers, bearing both zwitterionic phosphorylcholine groups and hydrophobic butyl side chains (PMB) and additional cross-linkable trimethoxysilylpropyl side chains (PMBT), were synthesized and coated on PLA NPs. Effects of the zwitterionic copolymer coatings on the NP size distribution, dispersion stability, and drug release behavior were investigated. Furthermore, the effect of the coatings on phagocytosis was also investigated. Compared with conventional polyvinyl alcohol coating, the cell membrane mimetic copolymer coatings decreased the size and increased the stability of the PLA NPs aqueous dispersions. More importantly, doxorubicin (DOX) release was well controlled and NPs phagocytosis by mouse peritoneal macrophage was decreased to one-third when the nanoparticles were coated with PMBT. This simple and effective zwitterionic polymer coating strategy may serve as a new route to design and optimize long-circulating intravenously injectable nanoparticle drug carriers. Keywords: poly(lactic acid); nanoparticle; zwitterion; phosphorylcholine; surface modification; phagocytosis; cell membrane mimetic polymer
1. Introduction Poly(lactic acid) (PLA) is a FDA-approved biodegradable, bioabsorbable, and biocompatible polymer which is broken down to monomeric units of lactic acid in the body. PLA nanoparticles are considered as one of the most promising polymer nanoparticles for drug delivery and targeting.[1] However, the nanoparticles administered intravenously are recognized as foreign bodies and are rapidly cleared from the body by phagocytic cells. Thus, the therapeutic effect of drugs delivered via nanoparticles is minimized and the toxicity of the drugs can be intolerable.[2,3] In order for the nanoparticles to escape the mononuclear phagocytic system (MPS) recognition and *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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subsequent clearance, the surface modification of nanoparticles, therefore, plays a critical role.[4,5] Hydrophilic polymers such as polyethylene glycol (PEG), poloxamers, and polysorbate and polysaccharides like dextran have been used to coat conventional nanoparticles to change the surface properties of nanoparticles.[6–11] These polymer coatings provide a dynamic cloud of hydrophilic and neutral chains at the particle surface, which repels plasma proteins. PEG and PEG-containing copolymer adsorption or grafting to the surface of nanoparticles is the most preferred method of surface modification.[5,12,13] The addition of PEG chains to the surface of nanoparticles results in an increase in the blood circulation half-life of the nanoparticles. Although the exact mechanisms by which PEG-prolonged circulation time of the surface modified nanoparticles is still not well understood, it is believed that the increased half-life of the nanoparticles in the blood is mainly due to prevention of opsonization of nanoparticles by plasma proteins (opsonins).[5] The hydrated PEG chains coated on the nanoparticle surfaces cause steric repulsion to the approach of opsonins. Although PEGylation has been extensively used on a variety of nanoparticle systems to increase surface hydrophilicity and improve circulation half-life by decreasing the interactions of blood proteins and MPS cells,[14–17] reported rapid clearance of PEGylated nanoparticles from blood, upon repeated injections, is still a serious problem.[18–20] It is reported that PLA nanoparticles immobilized with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer have the potential to improve blood compatibility and use as a novel drug carrier and diagnostic reagent,[21–23] since phosphorylcholine is the hydrophilic head group of cell outer membrane. Nanoparticles coated with zwitterionic groups, especially phosphorylcholine groups, have been shown excellent hemocompatibility for potential long circulation time in bloodstream.[24,25] We have synthesized a series of amphiphilic random copolymers [26] comprised of MPC, butyl methacrylate (BMA), and/or trimethoxysilylpropyl methacrylate (TSMA), and established simple methods of surface modification for chitosan film [27] and polypropylene hollow fiber membrane.[28] As the amphiphilic copolymers bear zwitterionic phosphorylcholine groups and hydrophobic butyl side chains, they can adsorb onto a hydrophobic surface from aqueous solution to form a cell outer membrane mimetic coating. Such a cell membrane mimetic coating shows excellent resistance to protein adsorption and platelet adhesion.[27–30] Here, we report for the first time the surface modification of PLA nanoparticles with cell membrane mimetic and cross-linkable random copolymers. The results suggest that nanoparticles coated with cell membrane mimetic and cross-linkable copolymer increased stability of the nanoparticles’ aqueous dispersion and decreased the macrophage clearance significantly. This simple and effective surface modification method may serve as a new route to design and optimize long-circulating and effective targeting drug nanocarriers. 2. Materials and methods 2.1. Materials MPC was synthesized according to the method reported by Ishihara et al. [31]. BMA, methacryloxyethyltrimethyl ammonium chloride, and 3-(TSMA) were purchased from Sigma-Aldrich Co. 2,2′-Azoisobutyronitrile was recrystallized from methanol and dried in vacuum. 6-Coumarin of laser grade was purchased from J & K Reagent Company. L929 cells were obtained from the Fourth Military Medical University of China.
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Bal b/c mice of 6–8-week-old were obtained from Animal Center of Xi’an Jiaotong University. Deionized water was purified by a Millipore system (Direct-Q3UV, France). Cell membrane mimetic and amphiphilic copolymers PMB and cross-linkable terpolymers PMBT were synthesized according to previous report.[32] Briefly, MPC as the hydrophilic monomer, BMA as the hydrophobic monomer, and TSMA as the crosslinkable monomer were copolymerized by free-radical polymerization through ‘monomer-starved’ approach.[33] The molar ratio of monomer units in the copolymers PMB and PMBT were determined by 1H NMR spectroscopy with an Inova 400 Hz NMR spectrometer (Varian, America). The PMB polymer contains 40% MPC mole unit and the PMBT contains 12% TSMA and 40% MPC mole units. MALDI TOF mass spectra showed m/z peaks at 6600–7200, suggesting that the average molecular weights of PMB and PMBT were greater than 6600 Da. The polymer structural formulas are shown in Figure 1. 2.2. Preparation of PLA nanoparticles PLA nanoparticles were prepared by a modified nanoprecipitation technique.[34] Briefly, PLA polymer was dissolved in acetone to form a solution of 5–15 mg/ml, followed by precipitation under vigorous agitation in water containing polyvinyl alcohol (PVA), PMB, or PMBT polymer as the stabilizer. After being stirred for two days to evaporate the organic solvent, the nanoparticles were separated by centrifugation at 12,000 rpm, and then redispersed in pure water. The content of the PLA nanoparticles in the water dispersions was from 0.2 to 1.0 mg/ml. DOX and 6-coumarin-loaded PLA nanoparticles were prepared similarly. Hydrophobic DOX or 6-coumarin was firstly dissolved in dichloromethane, and then mixed with the above PLA polymer solution. Finally, the PLA polymer solution containing DOX or 6-coumarin was dropped in agitated water with PVA, PMB, or PMBT polymer as the stabilizer. After being stirred for two days to evaporate the organic solvent, the nanoparticles were separated by centrifugation at 12,000 rpm, and then redispersed in water.
x O
O
O
y O
O
P
O O
O
N H3C
CH3 CH3 PMB
Figure 1.
O
O
O
O
O P
z O
y O
x O
Si
OCH3 H3CO OCH3
O O
N CH3 H3C CH3 PMBT
Structures of the amphiphilic random copolymers PMB and PMBT.
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2.3. Characterization of the nanoparticles Particle size and polydispersity index of the nanoparticle aqueous dispersions were determined at 25 °C under an angle of 173° by Dynamic Light Scattering (DLS) using a Malvern Zetasizer Nano-ZS90 instrument. Morphology of the nanoparticles was observed with an atomic force microscope (MultiMode 8, Bruker Nano Inc.). The sample was prepared by drop coating 50 μl 0.2 mg/ml nanoparticles dispersion on a 3.2 cm2 glass substrate. The coating was dried firstly at room temperature and then heated at 100 °C for 1 h to form a closely packed nanoparticle film which completely covered the substrate surface. PMB and PMBT polymers coated on the nanoparticles were analyzed with an X-ray photoelectron spectroscope (XPS, Axis Ultra, Kratos instrument, UK). Preparation of the samples was carried out in the same way as that of the AFM observation. Stability of the nanoparticle aqueous dispersions was tested by DLS and precipitation observation. The nanoparticle aqueous dispersions were stored at room temperature for 30 days, and measured the change of particle size and precipitation amount. Hydrophilicity of the nanoparticles was tested by static contact angle measurement using a video-based OCA 20 contact angle goniometer (Dataphysics, Germany). The nanoparticle dispersions were drop-coated on glass substrates to form a closely packed nanoparticle layer which completely covered the substrate surface. After heating at 80 °C for 2 h and washing with sufficient water, the nanoparticle-coated substrates were tested by OCA 20 contact angle goniometer. Reported static contact angles were the average of at least three measurements. 2.4. In vitro phagocytic uptake tests Mouse peritoneal macrophage (MPM) cells were seeded in 24-well culture plates at 1.0 × 106 cells per milliliter in RPMI 1640 medium containing 10% FBS, followed by incubation in 5% CO2 incubator at 37 °C for 24 h to allow for cell adherence. After replacing the culture medium with 1000 μl of 6-coumarin-loaded nanoparticles dispersion (0.2 mg/ml) in culture medium, the cells were incubated at 37 °C for 2 h. After the medium was sucked out, and the adhered macrophages were rinsed thrice with PBS to remove the non-phagocytized nanoparticles, the cells were observed using a fluorescent microscope (Olympus IX71). For quantification analysis, the sucked-out medium after MPM uptake was centrifuged at 1500 rpm for 10 min, and the supernatant was measured with a fluorescent spectrophotometer to read the intensity of the fluorescence emission peak at 500 nm (λex = 430 nm). The macrophage uptake ratio was calculated as follows: Uptake ratio ¼ I0II 100%, where I0 and I are the fluorescence intensity of 0 the culture medium before and after the macrophage uptake, respectively. 2.5. Cytotoxicity measurement Cytotoxicity of the nanoparticles was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) using mouse connective tissue fibroblast cells (L929). L929 at 1.0 × 104 cells per milliliter in DMEM medium containing 10% FBS was seeded in 96-well culture plates, followed by incubation in 5% CO2 incubator at 37 °C for 24 h to allow for cell adherence. Culture medium was replaced with 100 μl of the nanoparticles/culture medium suspensions with different concentration of 0.05, 0.2, and 0.8 mg/ml. L929 cells without nanoparticles in the medium were used as
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control. After 24 h incubation, 20 μl of MTT solution (5 mg/ml) was added into each well and allowed to react for 4 h at 37 °C. Then the solution was removed and 150 μl of dimethylsulfoxide (DMSO) was added into each well and the plate was incubated for 10 min in order to dissolve the formazan crystals. The optical densities in each well were measured at 490 nm, and the cell viability was calculated by the following formula:
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Cell viabilityð%Þ ¼ ðODsample =ODcontrol Þ 100%
2.6. Evaluation of DOX release from PLA nanoparticles The release of the loaded DOX from the nanoparticles was examined by dialysis through a membrane (Spectra/por membrane tubing; MWCO 6–8 kDa, Spectrum Labs, CA, USA). The equilibrated concentration of DOX in the dialysate of varying pH values (100 mM acetate buffer pH 4.0; 100 mM phosphate buffer saline, pH 7.4) was determined by fluorescence spectrophotometry (Hitachi F-4500, Japan) at different time intervals. The free DOX released into the dialysate was plotted as a function of time to determine the drug release kinetics. 2.7. Statistical analysis All data were generated in three or six independent experiments. Results were expressed as mean ± SD. Data were analyzed with student’s t-test. The difference between treatments was considered to be significant at a level of p < 0.05. 3. Results and discussion 3.1. Preparation and stability of phospholipid polymer-coated PLA nanoparticles The two phospholipid polymers (PMB and PMBT), shown in Figure 1, bear both hydrophilic phosphorylcholine groups and hydrophobic butyl side chains. Since zwitterionic phosphorylcholine is the hydrophilic group of cell outer membrane phosphatidylcholine, the amphiphilic copolymers PMB and PMBT are called cell membrane mimetic polymers. When PLA polymer solution was dropped in water containing PMB or PMBT polymer as the stabilizer, the hydrophobic butyl side chains interacted with the hydrophobic PLA particle surface, and the phosphorylcholine groups of the cell membrane mimetic polymer were immobilized on the surface/interface of precipitated PLA nanoparticles. PLA nanoparticles prepared in the stabilizing polymer solutions of PVA, PMB, and PMBT are indicated as PLA–PVA, PLA–PMB, and PLA–PMBT, respectively. As the PMB and PMBT polymers contain 40% mol fraction of the hydrophilic phosphorylcholine monomer units determined by 1H NMR,[28] they are watersoluble in the nanoparticle preparation. The possibly excess amount of the stabilizing polymer was removed by centrifugation separation in the preparation process. The XPS spectra of the PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticle films are shown in Figure 2. In the case of PLA–PMB and PLA–PMBT nanoparticle films, both the phosphorous peak at 133 eV and nitrogen peak at 402 eV were observed clearly, indicating effective immobilization of the phospholipid polymer on the PLA nanoparticles.
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PLA-PMBT PLA-PMB PLA-PVA
Intensity (kcps)
200
160
120
80
0 800
600
400
200
0
Binding Energy (eV)
Figure 2. XPS spectra of the PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticle films. The inserts are the high-resolution spectra of N1s and P2p. The appearance of both N1s and P2p peaks on the PLA–PMB and PLA–PMBT nanoparticle films confirm the existence of PMB and PMBT polymers on the particle surfaces.
The PLA nanoparticles coated with phospholipid polymers formed a cell membrane mimetic polymer layer, and their physicochemical and biological properties could be changed remarkably. As shown in Figure 3 and Table 1, PMB- and PMBT-stabilized/ coated PLA nanoparticles decreased their particle size significantly compared with the PVA stabilized PLA nanoparticles. The number-averaged size decreased from 179.4 nm (PLA–PVA) to 119.6 and 103.1 nm for PLA–PMB and PLA–PMBT nanoparticles, respectively. The mean sizes of the PLA–PMB and PLA–PMBT nanoparticles are much smaller than that prepared by Konno et al. [21] and more preferable as long-circulating drug carriers. Meanwhile, the negative zeta potential values of PLA–PMB and
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20
Number (%)
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PLA-PMBT
15
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Figure 3. Particle size distributions of PVA-, PMB-, and PMBT-coated PLA nanoparticles prepared in 0.10 mg/ml of PVA, PMB, and PMBT aqueous solutions, respectively.
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Table 1. Preparation and characteristics of PLA nanoparticles prepared by the same method with different stabilizing polymers. *
Particle size (nm)
Nanoparticle
PLA (mg)
PLA–PVA PLA–PMB PLA–PMBT
15 15 15
Stabilizer PVA (7 mg) PMB (7 mg) PMBT (7 mg)
Z-average
Num-average
PDI
ζ-Potential (mV)
214.9 171.5 166.1
179.4 119.6 103.1
0.058 0.085 0.103
–30.0 –26.1 –15.4
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*Measured by DLS at 20 °C within a pH range of 6.2–6.4.
PLA–PMBT nanoparticles increased obviously by changing the stabilizer from PVA to PMB and PMBT (Table 1). The zeta potential of the PLA nanoparticle surface was around −60 mV. After adsorption of the neutral stabilizing polymers, the zeta potential reduced significantly to less then −30 mV. The negatively charged nanoparticles covered by PMB and PMBT suggested that the physically adsorbed neutral polymer layer was thinner or looser than a chemically grafted PMBN layer on PLA particle surface.[23] The increased zeta potential values of PLA–PMB and PLA–PMBT nanoparticles, as well as the strong hydrophilicity of PMB and PMBT coatings, enhanced the water dispersion stability of the nanoparticles. As shown in Table 2, precipitation of all the nanoparticles in water dispersion was observed at different lengths of storage at room temperature. However, the time of appearing small amount of precipitation was extended and the increment of the precipitation was suppressed remarkably by stabilizing/coating PLA nanoparticles with PMB or PMBT polymers. In order to well understand the stability of the differently modified PLA nanoparticles, 3D AFM images were observed on surfaces of the PLA nanoparticle films. Figure 4 showed clearly that PVA-stabilized/coated PLA nanoparticles formed large aggregates by coalescing. Number-averaged diameter of the closely aggregated PLA– PVA particles shown in Figure 4(A) was approximately 160 nm, slightly smaller than the number-averaged hydrodynamic size 179 nm measured by DLS. On the other hand, PMB- and PMBT-stabilized/coated PLA nanoparticles did not show many large aggregates on their particle film surfaces, especially on PLA–PMBT particle film surface. The number-averaged diameters of both PLA–PMB and PLA–PMBT particles shown Table 2. Precipitation of 0.20 mg/ml PLA nanoparticle aqueous dispersion stabilized by PVA, PMB, and PMBT, respectively. Nanoparticles 3h 1 day 3 days 5 days 10 days 20 days 30 days
PLA–PVA
PLA–PMB
PLA–PMBT
− − − + ++ +++ +++
− − − − − + +
− − − − + + +
Note: − Non-sedimentation or observable precipitation; + Observable small amount of precipitation; +++ Obviously increased amount of precipitation.
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Figure 4. films.
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AFM 3D images of (A) PLA–PVA, (B) PLA–PMB, and (C) PLA–PMBT nanoparticle
in Figures 4(B) and (C) were smaller than 60 nm, which was much smaller than the corresponding number-averaged hydrodynamic sizes (119 and 103 nm) showed in Table 1. The greatly increased hydrodynamic diameters of PLA–PMB and PLA–PMBT nanoparticles suggested a thicker hydrated layer of PMB and PMBT coating than that of PVA. Therefore, the strongly hydrophilic PMB and PMBT polymer coating layer improved the stability of PLA–PMB and PLA–PMBT nanoparticle’s water dispersions. Furthermore, the hydrolysis and condensation/cross-linking of the trimethoxysilylpropyl groups (side chains) of PMBT polymers [24,25,30] increased the stability of the coating layer on PLA–PMBT nanoparticles. The cross-linked hydrophilic PMBT coating layer on PLA–PMBT nanoparticles completely prevented the particles to aggregate in the water as shown in Figure 4(C), resulting in the smallest hydrodynamic size (Figure 3). Water contact angle measurement was employed to provide further information on the stability of the PLA nanoparticle aqueous dispersions. As shown in Figure 5, the static water contact angle of PLA–PMB nanoparticle film surface was much smaller than that of PLA–PVA, suggesting more hydrophilic of PLA–PMB nanoparticle surface and more stable PLA–PMB nanoparticle water dispersion than that of PLA–PVA. On the other hand, the large static water contact angle of PLA–PMBT nanoparticle surface was attributed to cross-linking of the PMBT side chains.[28,33] It is well known that hydrophobic segments and side chains can migrate or reorientate to the surface of polymeric materials during drying in the air.[35] All the nanoparticle films shown in Figure 5 were prepared by evaporation and heating at 80 °C for 2 h, therefore the hydrophobic segments and side chains of the nanoparticles migrated to the particle surface to reduce free energy. During drying and heating, the cross-link density of the PMBT coating was increased and the hydrophobic surface structure was immobilized. Unlike the reversible surface structure of PMB film, the cross-linked PMBT nanoparticle film showed a large water contact angle.[33] Further experiments showed that all
Figure 5. Static water contact angles on (A) PLA–PVA, (B) PLA–PMB, and (C) PLA–PMBT nanoparticle films.
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the nanoparticle films prepared at room temperature had much smaller static water contact angles (42° for PLA–PMBT, 43° for PLA–PMB, and 52° for PLA–PVA film), suggesting strong hydrophilicity of PLA–PMBT and PLA–PMB nanoprticles. 3.2. In vitro phagocytic uptake
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PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticles loaded with 6-coumarin emit green fluorescence under the excitation wavelength of 430 nm. Phagocytes uptake of the fluorescent nanoparticles was observed with an inverted fluorescence microscope. As shown in Figure 6, all the MPM cells could be observed in the bright-field images, and only the cells that swallowed nanoparticles containing fluorescent probe could be
Figure 6. Fluorescence microscopic images demonstrating PLA–PVA, PLA–PMB, and PLA– PMBT nanoparticles phagocytized by MPMs for 2 h.
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observed in the fluorescent field images. As the three kinds of nanoparticles loaded with almost same concentration of 6-coumarin probes and the images were taken under exactly the same conditions, the brightness of the fluorescence images indicated the more cellular uptake of the nanoparticles. The fluorescence brightness of the MPM images was in the order of PLA–PVA > PLA–PMB > PLA–PMBT. In addition to visualization of the fluorescent images, the phagocytic uptake of the nanoparticles was also evaluated quantitatively by fluorescence spectrophotometry. The fluorescence intensities of the nanoparticle solutions before and after the cellular uptake were measured and the uptake percentages of the nanoparticles were calculated from changes of their fluorescence intensities. The results shown in Figure 7 were consistent with the observed images by inverted fluorescence microscope shown in Figure 6. The cellular uptake efficiency of PLA–PVA nanoparticles was three times higher than that of PLA–PMBT nanoparticles, and two times higher than that of PLA–PMB nanoparticles. It was reported that the uptake of nanoparticles by the MPS in vivo was closely related to the surface properties and opsonization, the stronger the surface hydrophobicity, the more easily it can be recognized and cleared by the MPS.[36] The improved hydrophilicity and the significant reduction in phagocytic uptake of the PLA–PMB and PLA–PMBT nanoparticles suggest that the PMB and PMBT zwitterionic phospholipid copolymer coating is a promising strategy to improve the physicochemical and biological properties of PLA nanoparticles. On the other hand, to prevent protein opsonization, the PMB zwitterionic phospholipid copolymer coating could not completely suppress the uptake of the particles by MPS which might be due to the too thin or too loose coating layer. For example, bovine serum albumin protein adsorption amount on the PMB-modified PLA particles (0.81 μg/mg) was much higher than that of PMBNcovered PLA particles (0.096 μg/mg).[23] 3.3. Cytotoxicity of the PLA nanoparticles L929 cells were chosen as an in vitro model to assess the cytotoxicity of the PLA nanoparticles. For comparison, a positively charged polymer micelle (PDS) was prepared using the copolymer of methacryloxyethyltrimethyl ammonium chloride and stearyl methacrylate. L929 cells were incubated with the nanoparticles for 48 h at
1000
Flourescence Intensity
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800 600 400 200 0 PLA-PVA
PLA-PMB
PLA-PMBT
Figure 7. Fluorescence intensities of PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticles phagocytized by MPMs for 2 h.
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3.4. Drug release of the PLA nanoparticles DOX is a widely used antineoplastic agent. DOX-loaded nanoparticles has led to its controlled release over extended periods of time, thereby increasing its efficacy and reducing toxic side effects.[38] Since the extracellular pH of solid tumors is signifi-
PLA-PVA PLA-PMB PLA-PMBT PDS
Cell Viability (%)
100 80 60 40 20 0 0.05 mg/mI
0.20mg/ml
0.80mg/ml
Figure 8. Cytotoxicity of different polymeric nanoparticles analyzed by MTT assay. L929 cells were cultured with different concentrations of the nanoparticles for 48 h. Data represent mean ± SD, n = 4.
60
Cumulative Release (%)
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graded concentrations of 0.05, 0.20, and 0.80 mg/ml. As shown in Figure 8, the cytotoxicities of all the PLA nanoparticles were significantly lower than that of the positively charged PDS micelles, indicating no significant cytotoxicity even at high concentration of 0.20 mg/ml for 48 h. The low cytotoxicity of the PLA nanoparticles was comparable to well-investigated PLGA-mPEG nanoparticles,[37] suggesting their suitability as drug carriers.
PLA-PVA PLA-PMB PLA-PMBT
50 40 30 20 10 0 0
10
20
30
40
50
60
Time (h)
Figure 9. DOX release profiles from PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticles upon incubation at pH 4.0 and 37 °C.
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cantly more acidic than that of normal tissues, drug release profiles of the DOX-loaded PLA nanoparticles were evaluated in buffers of both pH 4 and 7.4. As shown in Figure 9, the conventional PLA–PVA nanoparticle released the drug much faster than PLA–PMB and PLA–PMBT. PLA–PMBT nanoparticle showed the lowest rate of DOX release in the buffered aqueous solution of pH 4. The greatly decreased release rates of DOX in the PLA–PMB and PLA–PMBT nanoparticles suggested the success and effectiveness of the PLA nanoparticle surface modification with cell membrane mimetic polymers. The well-controlled release profiles from PLA–PMB and PLA–PMBT nanoparticles could be attributed to the effective interaction of PMB and PMBT amphiphilic polymers with the PLA particle surfaces. The strongly adsorbed PMB and PMBT hydrophilic layers not only stabilized the PLA–PMB and PLA–PMBT nanoparticles and masked the particles by forming ‘stealth’ coating to avoid phagocytosis, but also obstructed the diffusion of the loaded hydrophobic drug in the particles. 4. Conclusions Poly(lactic acid) nanoparticles were modified by both cross-linkable amphiphilic phospholipid polymer (PMBT) and non-cross-linkable amphiphilic phospholipid polymer (PMB). Both of the zwitterionic polymers formed a cell membrane mimetic coating on the PLA nanoparticle surface. Compared with conventional stabilizer PVA, PMBT and PMB aqueous solutions stabilize the PLA nanoparticles more effectively, and nanoparticles of less than 100 nm can be prepared. The cross-linked PMBT coating on the PLA nanoparticle surfaces can remarkably prevent the aggregation and phagocytosis of the coated particles. Furthermore, the antineoplastic drug DOX release is well controlled from the nanoparticles coated with PMBT. This simple and effective zwitterionic polymer-coating strategy may serve as a new route to design and optimize longcirculating controlled release nanoparticle drug carriers. Funding This work was supported by the National Natural Science Foundation of China [grant number 21244001], [grant number 21374087]; Japan Science Society [grant number S11-001] and the Fund of Engineering Laboratory of Xi’an City.
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[7] Martin-Banderas L, Duran-Lobato M, Munoz-Rubio I, Alvarez-Fuentes J, Fernandez-Arevalo M, Holgado MA. Functional PLGA NPs for oral drug delivery: recent strategies and developments. Mini-Rev. Med. Chem. 2013;13(1):58–69. [8] Feng L, Zhu C, Yuan H, Liu L, Lv F, Wang S. Conjugated polymer nanoparticles: preparation, properties, functionalization and biological applications. Chem. Soc. Rev. 2013;42:6620–6633. [9] Patil GB, Surana SJ. Fabrication and statistical optimization of surface engineered PLGA nanoparticles for naso-brain delivery of ropinirole hydrochloride: in vitro-ex-vivo studies. J. Biomater. Sci. Polym. Ed. 2013;24:1740–1756. [10] Liu W, Wei J, Huo P, Lu Y, Chen Y, Wei Y. Synthesis and cytotoxicity of brefeldin A conjugated monomethoxy-poly(ethylene glycol)-b-poly(L-lactide) polymeric micelles. J. Biomater. Sci., Polym. Ed. 2013;24:986–998. [11] Ren T, Xu N, Cao C, Yuan W, Yu X, Chen J, Ren J. Preparation and therapeutic efficacy of polysorbate-80-coated amphotericin B/PLA-b-PEG nanoparticles. J. Biomater. Sci. Polym. Ed. 2009;20:1369–1380. [12] Silva JM, Videira M, Gaspar R, Préat V, Florindo HF. Immune system targeting by biodegradable nanoparticles for cancer vaccines. J. Controlled Release. 2013;168:179–199. [13] Vijayakumar MR, Muthu MS, Singh S. Copolymers of poly(lactic acid) and D-α-tocopheryl polyethylene glycol 1000 succinate-based nanomedicines: versatile multifunctional platforms for cancer diagnosis and therapy. Expert Opin. Drug Delivery. 2013;10:529–543. [14] Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistrubution of polymeric nanoparticles. Mol. Pharm. 2008;5:505–515. [15] Knop K, Hoogenboom R, Fischer D, Schubert US. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem. Int. Ed. 2010;49:6288–6308. [16] Mosqueira VCF, Legrand P, Morgat JL, Vert M, Mysiakine E, Gref R, Devissaguet JP, Barrett G. Biodistribution of long-circulating PEG-grafted nanocapsules in mice: effects of PEG chain length and density. Pharm. Res. 2001;18:1411–1419. [17] Moghimi SM, Szebeni J. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonization and protein-binding properties. Prog. Lipid Res. 2003;42:463–478. [18] Ishida T, Maeda R, Ichihara M, Irimura K, Kiwada H. Accelerated clearance of PEGylated liposomes in rats after repeated injections. J. Controlled Release. 2003;88:35–42. [19] Ishida T, Masuda K, Ichikawa T, Ichihara M, Irimura K, Kiwada H. Accelerated clearance of a second injection of PEGylated liposomes in mice. Int. J. Pharm. 2003;255:167–174. [20] Dams ETM, Laverman P, Oyen WJG, Storm G, Scherphof GL, van der Meer JWM, et al. Accelerated blood clearance and altered biodistribution of repeated injections of sterically stabilized liposomes. J. Pharmacol. Exp. Ther. 2000;292:1071–1079. [21] Konno T, Kurita K, Iwasaki Y, Nakabayashi N, Ishihara K. Preparation of nanoparticles composed with bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer. Biomaterials. 2001;22:1883–1889. [22] Matsuno R, Ishihara K. Integrated functional nanocolloids covered with artificial cell membranes for biomedical applications. Nano Today. 2011;6:61–74. [23] Ishihara K, Goto Y, Takai M, Matsuno R, Inoue Y, Konno T. Novel polymer biomaterials and interfaces inspired from cell membrane functions. Biochim. Biophys. Acta, Gen. Subj. 2011;1810:268–275. [24] Zeng R, Fu H, Zhao YF. Synthesis of novel biomimetic zwitterionic phosphorylcholinebound chitosan derivative. Macromol. Rapid Commun. 2006;27:548–552. [25] Wang HB, Xu FM, Wang Y, Liu XS, Jin Q, Ji J. pH-responsive and biodegradable polymeric micelles based on poly(β-amino ester)-graft-phosphorylcholine for doxorubicin delivery. Polym. Chem. 2013;4:3012–3019. [26] Zong M, Gong Y. Fabrication and biocompatibility of cell outer membrane mimetic surfaces. Chin. J. Polym. Sci. 2011;29:53–64. [27] Gong M, Dang Y, Wang Y, Yang S, Winnik FM, Gong Y. Cell membrane mimetic films immobilized by synergistic grafting and crosslinking. Soft Matter. 2013;9:4501–4508. [28] Wang Y, Gong M, Yang S, Nakashima K, Gong Y. Hemocompatibility and film stability improvement of cross-linkable MPC copolymer coated polypropylene hollow fiber membrane. J. Membr. Sci. 2014;452:29–36.
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[29] Gong Y, Liu L, Messersmith PB. Doubly biomimetic catecholic phosphorylcholine copolymer: a platform strategy for fabricating antifoul-ing surfaces. Macromol. Biosci. 2012;12:979–985. [30] Gong Y, Winnik FM. Strategies in biomimetic surface engineering of nanoparticles for biomedical applications. Nanoscale. 2012;4:360–368. [31] Ishihara K, Ueda T, Nakabayashi N. Preparation of phospholipid polymers and their properties as polymer hydrogel membranes. Polym. J. 1990;22:355–360. [32] Zhang J, Gong M, Yang S, Gong Y. Crosslinked biomimetic random copolymer micelles as potential anti-cancer drug delivery vehicle. J. Controlled Release. 2011;152:e23–e25. [33] Gong M, Yang S, Ma J, Zhang S, Winnik FM, Gong Y. Tunable cell membrane mimetic surfaces prepared with a novel phospholipid polymer. Appl. Surf. Sci. 2008;255:555–558. [34] Fessi H, Puisieux F, Devissaguet JP, Ammoury N, Benita S. Nanocapsule formation by interfacial polymer deposition following solvent displacement. Int. J. Pharm. 1989;55: R1–R4. [35] Yang S, Zhang S, Winnik FM, Mwale F, Gong Y. Group reorientation and migration of amphiphilic polymer bearing phosphorylcholine functionalities on surface of cellular membrane mimicking coating. J. Biomed. Mater. Res. Part A. 2008;84A:837–841. [36] Wilhelm C, Gazeau F, Roger J, Pons JN, Bacri JC. Interaction of anionic superparamagnetic nanoparticles with cells: kinetic analyses of membrane adsorption and subsequent internalization. Langmuir. 2002;18:8148–8155. [37] Gryparis EC, Hatziapostolou M, Papadimitriou E, Avgoustakis K. Anticancer activity of cisplatin loaded PLGA-mPEG nanoparticles on LNCaP prostate cancer cells. Eur. J. Pharm. Biopharm. 2007;67:1–8. [38] Missirlis D, Kawamura R, Tirelli N, Hubbell JA. Doxorubicin encapsulation and diffusional release from stable, polymeric, hydrogel nanoparticles. Eur. J. Pharm. Sci. 2006;29:120–129.
Journal of Biomaterials Science, Polymer Edition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsp20
Preparation and characterization of zwitterionic phospholipid polymercoated poly(lactic acid) nanoparticles a
a
ab
c
Li-Li Bao , Hao-Qiang Huang , Jing Zhao , Kenichi Nakashima & ad
Yong-Kuan Gong a
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, Shaanxi, P.R. China b
School of Environmental and Chemical Engineering, Xi’an Polytechnic University, Xi’an 710048, Shaanxi, P.R. China c
Faculty of Science and Engineering, Department of Chemistry, Saga University, 1 Honjo-machi, Saga 840-8502, Japan d
Engineering Laboratory for Bionic Biomaterials and Devices of Xi’an City, Northwest University, Xi’an 710069, Shaanxi, P.R. China Published online: 03 Sep 2014.
To cite this article: Li-Li Bao, Hao-Qiang Huang, Jing Zhao, Kenichi Nakashima & Yong-Kuan Gong (2014) Preparation and characterization of zwitterionic phospholipid polymer-coated poly(lactic acid) nanoparticles, Journal of Biomaterials Science, Polymer Edition, 25:14-15, 1703-1716, DOI: 10.1080/09205063.2014.952993 To link to this article: http://dx.doi.org/10.1080/09205063.2014.952993
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Journal of Biomaterials Science, Polymer Edition, 2014 Vol. 25, Nos. 14–15, 1703–1716, http://dx.doi.org/10.1080/09205063.2014.952993
Preparation and characterization of zwitterionic phospholipid polymer-coated poly(lactic acid) nanoparticles
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Li-Li Baoa, Hao-Qiang Huanga, Jing Zhaoa,b, Kenichi Nakashimac and Yong-Kuan Gonga,d* a Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, Shaanxi, P.R. China; bSchool of Environmental and Chemical Engineering, Xi’an Polytechnic University, Xi’an 710048, Shaanxi, P.R. China; cFaculty of Science and Engineering, Department of Chemistry, Saga University, 1 Honjo-machi, Saga 840-8502, Japan; dEngineering Laboratory for Bionic Biomaterials and Devices of Xi’an City, Northwest University, Xi’an 710069, Shaanxi, P.R. China
(Received 30 April 2014; accepted 6 August 2014) Poly(lactic acid) (PLA) nanoparticles (NPs) are the most promising polymer NPs for drug delivery and targeting. However, they are easily recognized as a foreign body and rapidly cleared from the body by the mononuclear phagocyte system. Cell membrane mimetic random copolymers, bearing both zwitterionic phosphorylcholine groups and hydrophobic butyl side chains (PMB) and additional cross-linkable trimethoxysilylpropyl side chains (PMBT), were synthesized and coated on PLA NPs. Effects of the zwitterionic copolymer coatings on the NP size distribution, dispersion stability, and drug release behavior were investigated. Furthermore, the effect of the coatings on phagocytosis was also investigated. Compared with conventional polyvinyl alcohol coating, the cell membrane mimetic copolymer coatings decreased the size and increased the stability of the PLA NPs aqueous dispersions. More importantly, doxorubicin (DOX) release was well controlled and NPs phagocytosis by mouse peritoneal macrophage was decreased to one-third when the nanoparticles were coated with PMBT. This simple and effective zwitterionic polymer coating strategy may serve as a new route to design and optimize long-circulating intravenously injectable nanoparticle drug carriers. Keywords: poly(lactic acid); nanoparticle; zwitterion; phosphorylcholine; surface modification; phagocytosis; cell membrane mimetic polymer
1. Introduction Poly(lactic acid) (PLA) is a FDA-approved biodegradable, bioabsorbable, and biocompatible polymer which is broken down to monomeric units of lactic acid in the body. PLA nanoparticles are considered as one of the most promising polymer nanoparticles for drug delivery and targeting.[1] However, the nanoparticles administered intravenously are recognized as foreign bodies and are rapidly cleared from the body by phagocytic cells. Thus, the therapeutic effect of drugs delivered via nanoparticles is minimized and the toxicity of the drugs can be intolerable.[2,3] In order for the nanoparticles to escape the mononuclear phagocytic system (MPS) recognition and *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
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subsequent clearance, the surface modification of nanoparticles, therefore, plays a critical role.[4,5] Hydrophilic polymers such as polyethylene glycol (PEG), poloxamers, and polysorbate and polysaccharides like dextran have been used to coat conventional nanoparticles to change the surface properties of nanoparticles.[6–11] These polymer coatings provide a dynamic cloud of hydrophilic and neutral chains at the particle surface, which repels plasma proteins. PEG and PEG-containing copolymer adsorption or grafting to the surface of nanoparticles is the most preferred method of surface modification.[5,12,13] The addition of PEG chains to the surface of nanoparticles results in an increase in the blood circulation half-life of the nanoparticles. Although the exact mechanisms by which PEG-prolonged circulation time of the surface modified nanoparticles is still not well understood, it is believed that the increased half-life of the nanoparticles in the blood is mainly due to prevention of opsonization of nanoparticles by plasma proteins (opsonins).[5] The hydrated PEG chains coated on the nanoparticle surfaces cause steric repulsion to the approach of opsonins. Although PEGylation has been extensively used on a variety of nanoparticle systems to increase surface hydrophilicity and improve circulation half-life by decreasing the interactions of blood proteins and MPS cells,[14–17] reported rapid clearance of PEGylated nanoparticles from blood, upon repeated injections, is still a serious problem.[18–20] It is reported that PLA nanoparticles immobilized with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer have the potential to improve blood compatibility and use as a novel drug carrier and diagnostic reagent,[21–23] since phosphorylcholine is the hydrophilic head group of cell outer membrane. Nanoparticles coated with zwitterionic groups, especially phosphorylcholine groups, have been shown excellent hemocompatibility for potential long circulation time in bloodstream.[24,25] We have synthesized a series of amphiphilic random copolymers [26] comprised of MPC, butyl methacrylate (BMA), and/or trimethoxysilylpropyl methacrylate (TSMA), and established simple methods of surface modification for chitosan film [27] and polypropylene hollow fiber membrane.[28] As the amphiphilic copolymers bear zwitterionic phosphorylcholine groups and hydrophobic butyl side chains, they can adsorb onto a hydrophobic surface from aqueous solution to form a cell outer membrane mimetic coating. Such a cell membrane mimetic coating shows excellent resistance to protein adsorption and platelet adhesion.[27–30] Here, we report for the first time the surface modification of PLA nanoparticles with cell membrane mimetic and cross-linkable random copolymers. The results suggest that nanoparticles coated with cell membrane mimetic and cross-linkable copolymer increased stability of the nanoparticles’ aqueous dispersion and decreased the macrophage clearance significantly. This simple and effective surface modification method may serve as a new route to design and optimize long-circulating and effective targeting drug nanocarriers. 2. Materials and methods 2.1. Materials MPC was synthesized according to the method reported by Ishihara et al. [31]. BMA, methacryloxyethyltrimethyl ammonium chloride, and 3-(TSMA) were purchased from Sigma-Aldrich Co. 2,2′-Azoisobutyronitrile was recrystallized from methanol and dried in vacuum. 6-Coumarin of laser grade was purchased from J & K Reagent Company. L929 cells were obtained from the Fourth Military Medical University of China.
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Bal b/c mice of 6–8-week-old were obtained from Animal Center of Xi’an Jiaotong University. Deionized water was purified by a Millipore system (Direct-Q3UV, France). Cell membrane mimetic and amphiphilic copolymers PMB and cross-linkable terpolymers PMBT were synthesized according to previous report.[32] Briefly, MPC as the hydrophilic monomer, BMA as the hydrophobic monomer, and TSMA as the crosslinkable monomer were copolymerized by free-radical polymerization through ‘monomer-starved’ approach.[33] The molar ratio of monomer units in the copolymers PMB and PMBT were determined by 1H NMR spectroscopy with an Inova 400 Hz NMR spectrometer (Varian, America). The PMB polymer contains 40% MPC mole unit and the PMBT contains 12% TSMA and 40% MPC mole units. MALDI TOF mass spectra showed m/z peaks at 6600–7200, suggesting that the average molecular weights of PMB and PMBT were greater than 6600 Da. The polymer structural formulas are shown in Figure 1. 2.2. Preparation of PLA nanoparticles PLA nanoparticles were prepared by a modified nanoprecipitation technique.[34] Briefly, PLA polymer was dissolved in acetone to form a solution of 5–15 mg/ml, followed by precipitation under vigorous agitation in water containing polyvinyl alcohol (PVA), PMB, or PMBT polymer as the stabilizer. After being stirred for two days to evaporate the organic solvent, the nanoparticles were separated by centrifugation at 12,000 rpm, and then redispersed in pure water. The content of the PLA nanoparticles in the water dispersions was from 0.2 to 1.0 mg/ml. DOX and 6-coumarin-loaded PLA nanoparticles were prepared similarly. Hydrophobic DOX or 6-coumarin was firstly dissolved in dichloromethane, and then mixed with the above PLA polymer solution. Finally, the PLA polymer solution containing DOX or 6-coumarin was dropped in agitated water with PVA, PMB, or PMBT polymer as the stabilizer. After being stirred for two days to evaporate the organic solvent, the nanoparticles were separated by centrifugation at 12,000 rpm, and then redispersed in water.
x O
O
O
y O
O
P
O O
O
N H3C
CH3 CH3 PMB
Figure 1.
O
O
O
O
O P
z O
y O
x O
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OCH3 H3CO OCH3
O O
N CH3 H3C CH3 PMBT
Structures of the amphiphilic random copolymers PMB and PMBT.
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2.3. Characterization of the nanoparticles Particle size and polydispersity index of the nanoparticle aqueous dispersions were determined at 25 °C under an angle of 173° by Dynamic Light Scattering (DLS) using a Malvern Zetasizer Nano-ZS90 instrument. Morphology of the nanoparticles was observed with an atomic force microscope (MultiMode 8, Bruker Nano Inc.). The sample was prepared by drop coating 50 μl 0.2 mg/ml nanoparticles dispersion on a 3.2 cm2 glass substrate. The coating was dried firstly at room temperature and then heated at 100 °C for 1 h to form a closely packed nanoparticle film which completely covered the substrate surface. PMB and PMBT polymers coated on the nanoparticles were analyzed with an X-ray photoelectron spectroscope (XPS, Axis Ultra, Kratos instrument, UK). Preparation of the samples was carried out in the same way as that of the AFM observation. Stability of the nanoparticle aqueous dispersions was tested by DLS and precipitation observation. The nanoparticle aqueous dispersions were stored at room temperature for 30 days, and measured the change of particle size and precipitation amount. Hydrophilicity of the nanoparticles was tested by static contact angle measurement using a video-based OCA 20 contact angle goniometer (Dataphysics, Germany). The nanoparticle dispersions were drop-coated on glass substrates to form a closely packed nanoparticle layer which completely covered the substrate surface. After heating at 80 °C for 2 h and washing with sufficient water, the nanoparticle-coated substrates were tested by OCA 20 contact angle goniometer. Reported static contact angles were the average of at least three measurements. 2.4. In vitro phagocytic uptake tests Mouse peritoneal macrophage (MPM) cells were seeded in 24-well culture plates at 1.0 × 106 cells per milliliter in RPMI 1640 medium containing 10% FBS, followed by incubation in 5% CO2 incubator at 37 °C for 24 h to allow for cell adherence. After replacing the culture medium with 1000 μl of 6-coumarin-loaded nanoparticles dispersion (0.2 mg/ml) in culture medium, the cells were incubated at 37 °C for 2 h. After the medium was sucked out, and the adhered macrophages were rinsed thrice with PBS to remove the non-phagocytized nanoparticles, the cells were observed using a fluorescent microscope (Olympus IX71). For quantification analysis, the sucked-out medium after MPM uptake was centrifuged at 1500 rpm for 10 min, and the supernatant was measured with a fluorescent spectrophotometer to read the intensity of the fluorescence emission peak at 500 nm (λex = 430 nm). The macrophage uptake ratio was calculated as follows: Uptake ratio ¼ I0II 100%, where I0 and I are the fluorescence intensity of 0 the culture medium before and after the macrophage uptake, respectively. 2.5. Cytotoxicity measurement Cytotoxicity of the nanoparticles was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) using mouse connective tissue fibroblast cells (L929). L929 at 1.0 × 104 cells per milliliter in DMEM medium containing 10% FBS was seeded in 96-well culture plates, followed by incubation in 5% CO2 incubator at 37 °C for 24 h to allow for cell adherence. Culture medium was replaced with 100 μl of the nanoparticles/culture medium suspensions with different concentration of 0.05, 0.2, and 0.8 mg/ml. L929 cells without nanoparticles in the medium were used as
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control. After 24 h incubation, 20 μl of MTT solution (5 mg/ml) was added into each well and allowed to react for 4 h at 37 °C. Then the solution was removed and 150 μl of dimethylsulfoxide (DMSO) was added into each well and the plate was incubated for 10 min in order to dissolve the formazan crystals. The optical densities in each well were measured at 490 nm, and the cell viability was calculated by the following formula:
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Cell viabilityð%Þ ¼ ðODsample =ODcontrol Þ 100%
2.6. Evaluation of DOX release from PLA nanoparticles The release of the loaded DOX from the nanoparticles was examined by dialysis through a membrane (Spectra/por membrane tubing; MWCO 6–8 kDa, Spectrum Labs, CA, USA). The equilibrated concentration of DOX in the dialysate of varying pH values (100 mM acetate buffer pH 4.0; 100 mM phosphate buffer saline, pH 7.4) was determined by fluorescence spectrophotometry (Hitachi F-4500, Japan) at different time intervals. The free DOX released into the dialysate was plotted as a function of time to determine the drug release kinetics. 2.7. Statistical analysis All data were generated in three or six independent experiments. Results were expressed as mean ± SD. Data were analyzed with student’s t-test. The difference between treatments was considered to be significant at a level of p < 0.05. 3. Results and discussion 3.1. Preparation and stability of phospholipid polymer-coated PLA nanoparticles The two phospholipid polymers (PMB and PMBT), shown in Figure 1, bear both hydrophilic phosphorylcholine groups and hydrophobic butyl side chains. Since zwitterionic phosphorylcholine is the hydrophilic group of cell outer membrane phosphatidylcholine, the amphiphilic copolymers PMB and PMBT are called cell membrane mimetic polymers. When PLA polymer solution was dropped in water containing PMB or PMBT polymer as the stabilizer, the hydrophobic butyl side chains interacted with the hydrophobic PLA particle surface, and the phosphorylcholine groups of the cell membrane mimetic polymer were immobilized on the surface/interface of precipitated PLA nanoparticles. PLA nanoparticles prepared in the stabilizing polymer solutions of PVA, PMB, and PMBT are indicated as PLA–PVA, PLA–PMB, and PLA–PMBT, respectively. As the PMB and PMBT polymers contain 40% mol fraction of the hydrophilic phosphorylcholine monomer units determined by 1H NMR,[28] they are watersoluble in the nanoparticle preparation. The possibly excess amount of the stabilizing polymer was removed by centrifugation separation in the preparation process. The XPS spectra of the PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticle films are shown in Figure 2. In the case of PLA–PMB and PLA–PMBT nanoparticle films, both the phosphorous peak at 133 eV and nitrogen peak at 402 eV were observed clearly, indicating effective immobilization of the phospholipid polymer on the PLA nanoparticles.
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PLA-PMBT PLA-PMB PLA-PVA
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0 800
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0
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Figure 2. XPS spectra of the PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticle films. The inserts are the high-resolution spectra of N1s and P2p. The appearance of both N1s and P2p peaks on the PLA–PMB and PLA–PMBT nanoparticle films confirm the existence of PMB and PMBT polymers on the particle surfaces.
The PLA nanoparticles coated with phospholipid polymers formed a cell membrane mimetic polymer layer, and their physicochemical and biological properties could be changed remarkably. As shown in Figure 3 and Table 1, PMB- and PMBT-stabilized/ coated PLA nanoparticles decreased their particle size significantly compared with the PVA stabilized PLA nanoparticles. The number-averaged size decreased from 179.4 nm (PLA–PVA) to 119.6 and 103.1 nm for PLA–PMB and PLA–PMBT nanoparticles, respectively. The mean sizes of the PLA–PMB and PLA–PMBT nanoparticles are much smaller than that prepared by Konno et al. [21] and more preferable as long-circulating drug carriers. Meanwhile, the negative zeta potential values of PLA–PMB and
25
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Number (%)
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Figure 3. Particle size distributions of PVA-, PMB-, and PMBT-coated PLA nanoparticles prepared in 0.10 mg/ml of PVA, PMB, and PMBT aqueous solutions, respectively.
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Table 1. Preparation and characteristics of PLA nanoparticles prepared by the same method with different stabilizing polymers. *
Particle size (nm)
Nanoparticle
PLA (mg)
PLA–PVA PLA–PMB PLA–PMBT
15 15 15
Stabilizer PVA (7 mg) PMB (7 mg) PMBT (7 mg)
Z-average
Num-average
PDI
ζ-Potential (mV)
214.9 171.5 166.1
179.4 119.6 103.1
0.058 0.085 0.103
–30.0 –26.1 –15.4
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*Measured by DLS at 20 °C within a pH range of 6.2–6.4.
PLA–PMBT nanoparticles increased obviously by changing the stabilizer from PVA to PMB and PMBT (Table 1). The zeta potential of the PLA nanoparticle surface was around −60 mV. After adsorption of the neutral stabilizing polymers, the zeta potential reduced significantly to less then −30 mV. The negatively charged nanoparticles covered by PMB and PMBT suggested that the physically adsorbed neutral polymer layer was thinner or looser than a chemically grafted PMBN layer on PLA particle surface.[23] The increased zeta potential values of PLA–PMB and PLA–PMBT nanoparticles, as well as the strong hydrophilicity of PMB and PMBT coatings, enhanced the water dispersion stability of the nanoparticles. As shown in Table 2, precipitation of all the nanoparticles in water dispersion was observed at different lengths of storage at room temperature. However, the time of appearing small amount of precipitation was extended and the increment of the precipitation was suppressed remarkably by stabilizing/coating PLA nanoparticles with PMB or PMBT polymers. In order to well understand the stability of the differently modified PLA nanoparticles, 3D AFM images were observed on surfaces of the PLA nanoparticle films. Figure 4 showed clearly that PVA-stabilized/coated PLA nanoparticles formed large aggregates by coalescing. Number-averaged diameter of the closely aggregated PLA– PVA particles shown in Figure 4(A) was approximately 160 nm, slightly smaller than the number-averaged hydrodynamic size 179 nm measured by DLS. On the other hand, PMB- and PMBT-stabilized/coated PLA nanoparticles did not show many large aggregates on their particle film surfaces, especially on PLA–PMBT particle film surface. The number-averaged diameters of both PLA–PMB and PLA–PMBT particles shown Table 2. Precipitation of 0.20 mg/ml PLA nanoparticle aqueous dispersion stabilized by PVA, PMB, and PMBT, respectively. Nanoparticles 3h 1 day 3 days 5 days 10 days 20 days 30 days
PLA–PVA
PLA–PMB
PLA–PMBT
− − − + ++ +++ +++
− − − − − + +
− − − − + + +
Note: − Non-sedimentation or observable precipitation; + Observable small amount of precipitation; +++ Obviously increased amount of precipitation.
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Figure 4. films.
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AFM 3D images of (A) PLA–PVA, (B) PLA–PMB, and (C) PLA–PMBT nanoparticle
in Figures 4(B) and (C) were smaller than 60 nm, which was much smaller than the corresponding number-averaged hydrodynamic sizes (119 and 103 nm) showed in Table 1. The greatly increased hydrodynamic diameters of PLA–PMB and PLA–PMBT nanoparticles suggested a thicker hydrated layer of PMB and PMBT coating than that of PVA. Therefore, the strongly hydrophilic PMB and PMBT polymer coating layer improved the stability of PLA–PMB and PLA–PMBT nanoparticle’s water dispersions. Furthermore, the hydrolysis and condensation/cross-linking of the trimethoxysilylpropyl groups (side chains) of PMBT polymers [24,25,30] increased the stability of the coating layer on PLA–PMBT nanoparticles. The cross-linked hydrophilic PMBT coating layer on PLA–PMBT nanoparticles completely prevented the particles to aggregate in the water as shown in Figure 4(C), resulting in the smallest hydrodynamic size (Figure 3). Water contact angle measurement was employed to provide further information on the stability of the PLA nanoparticle aqueous dispersions. As shown in Figure 5, the static water contact angle of PLA–PMB nanoparticle film surface was much smaller than that of PLA–PVA, suggesting more hydrophilic of PLA–PMB nanoparticle surface and more stable PLA–PMB nanoparticle water dispersion than that of PLA–PVA. On the other hand, the large static water contact angle of PLA–PMBT nanoparticle surface was attributed to cross-linking of the PMBT side chains.[28,33] It is well known that hydrophobic segments and side chains can migrate or reorientate to the surface of polymeric materials during drying in the air.[35] All the nanoparticle films shown in Figure 5 were prepared by evaporation and heating at 80 °C for 2 h, therefore the hydrophobic segments and side chains of the nanoparticles migrated to the particle surface to reduce free energy. During drying and heating, the cross-link density of the PMBT coating was increased and the hydrophobic surface structure was immobilized. Unlike the reversible surface structure of PMB film, the cross-linked PMBT nanoparticle film showed a large water contact angle.[33] Further experiments showed that all
Figure 5. Static water contact angles on (A) PLA–PVA, (B) PLA–PMB, and (C) PLA–PMBT nanoparticle films.
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the nanoparticle films prepared at room temperature had much smaller static water contact angles (42° for PLA–PMBT, 43° for PLA–PMB, and 52° for PLA–PVA film), suggesting strong hydrophilicity of PLA–PMBT and PLA–PMB nanoprticles. 3.2. In vitro phagocytic uptake
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PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticles loaded with 6-coumarin emit green fluorescence under the excitation wavelength of 430 nm. Phagocytes uptake of the fluorescent nanoparticles was observed with an inverted fluorescence microscope. As shown in Figure 6, all the MPM cells could be observed in the bright-field images, and only the cells that swallowed nanoparticles containing fluorescent probe could be
Figure 6. Fluorescence microscopic images demonstrating PLA–PVA, PLA–PMB, and PLA– PMBT nanoparticles phagocytized by MPMs for 2 h.
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observed in the fluorescent field images. As the three kinds of nanoparticles loaded with almost same concentration of 6-coumarin probes and the images were taken under exactly the same conditions, the brightness of the fluorescence images indicated the more cellular uptake of the nanoparticles. The fluorescence brightness of the MPM images was in the order of PLA–PVA > PLA–PMB > PLA–PMBT. In addition to visualization of the fluorescent images, the phagocytic uptake of the nanoparticles was also evaluated quantitatively by fluorescence spectrophotometry. The fluorescence intensities of the nanoparticle solutions before and after the cellular uptake were measured and the uptake percentages of the nanoparticles were calculated from changes of their fluorescence intensities. The results shown in Figure 7 were consistent with the observed images by inverted fluorescence microscope shown in Figure 6. The cellular uptake efficiency of PLA–PVA nanoparticles was three times higher than that of PLA–PMBT nanoparticles, and two times higher than that of PLA–PMB nanoparticles. It was reported that the uptake of nanoparticles by the MPS in vivo was closely related to the surface properties and opsonization, the stronger the surface hydrophobicity, the more easily it can be recognized and cleared by the MPS.[36] The improved hydrophilicity and the significant reduction in phagocytic uptake of the PLA–PMB and PLA–PMBT nanoparticles suggest that the PMB and PMBT zwitterionic phospholipid copolymer coating is a promising strategy to improve the physicochemical and biological properties of PLA nanoparticles. On the other hand, to prevent protein opsonization, the PMB zwitterionic phospholipid copolymer coating could not completely suppress the uptake of the particles by MPS which might be due to the too thin or too loose coating layer. For example, bovine serum albumin protein adsorption amount on the PMB-modified PLA particles (0.81 μg/mg) was much higher than that of PMBNcovered PLA particles (0.096 μg/mg).[23] 3.3. Cytotoxicity of the PLA nanoparticles L929 cells were chosen as an in vitro model to assess the cytotoxicity of the PLA nanoparticles. For comparison, a positively charged polymer micelle (PDS) was prepared using the copolymer of methacryloxyethyltrimethyl ammonium chloride and stearyl methacrylate. L929 cells were incubated with the nanoparticles for 48 h at
1000
Flourescence Intensity
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800 600 400 200 0 PLA-PVA
PLA-PMB
PLA-PMBT
Figure 7. Fluorescence intensities of PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticles phagocytized by MPMs for 2 h.
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3.4. Drug release of the PLA nanoparticles DOX is a widely used antineoplastic agent. DOX-loaded nanoparticles has led to its controlled release over extended periods of time, thereby increasing its efficacy and reducing toxic side effects.[38] Since the extracellular pH of solid tumors is signifi-
PLA-PVA PLA-PMB PLA-PMBT PDS
Cell Viability (%)
100 80 60 40 20 0 0.05 mg/mI
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0.80mg/ml
Figure 8. Cytotoxicity of different polymeric nanoparticles analyzed by MTT assay. L929 cells were cultured with different concentrations of the nanoparticles for 48 h. Data represent mean ± SD, n = 4.
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Cumulative Release (%)
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graded concentrations of 0.05, 0.20, and 0.80 mg/ml. As shown in Figure 8, the cytotoxicities of all the PLA nanoparticles were significantly lower than that of the positively charged PDS micelles, indicating no significant cytotoxicity even at high concentration of 0.20 mg/ml for 48 h. The low cytotoxicity of the PLA nanoparticles was comparable to well-investigated PLGA-mPEG nanoparticles,[37] suggesting their suitability as drug carriers.
PLA-PVA PLA-PMB PLA-PMBT
50 40 30 20 10 0 0
10
20
30
40
50
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Figure 9. DOX release profiles from PLA–PVA, PLA–PMB, and PLA–PMBT nanoparticles upon incubation at pH 4.0 and 37 °C.
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cantly more acidic than that of normal tissues, drug release profiles of the DOX-loaded PLA nanoparticles were evaluated in buffers of both pH 4 and 7.4. As shown in Figure 9, the conventional PLA–PVA nanoparticle released the drug much faster than PLA–PMB and PLA–PMBT. PLA–PMBT nanoparticle showed the lowest rate of DOX release in the buffered aqueous solution of pH 4. The greatly decreased release rates of DOX in the PLA–PMB and PLA–PMBT nanoparticles suggested the success and effectiveness of the PLA nanoparticle surface modification with cell membrane mimetic polymers. The well-controlled release profiles from PLA–PMB and PLA–PMBT nanoparticles could be attributed to the effective interaction of PMB and PMBT amphiphilic polymers with the PLA particle surfaces. The strongly adsorbed PMB and PMBT hydrophilic layers not only stabilized the PLA–PMB and PLA–PMBT nanoparticles and masked the particles by forming ‘stealth’ coating to avoid phagocytosis, but also obstructed the diffusion of the loaded hydrophobic drug in the particles. 4. Conclusions Poly(lactic acid) nanoparticles were modified by both cross-linkable amphiphilic phospholipid polymer (PMBT) and non-cross-linkable amphiphilic phospholipid polymer (PMB). Both of the zwitterionic polymers formed a cell membrane mimetic coating on the PLA nanoparticle surface. Compared with conventional stabilizer PVA, PMBT and PMB aqueous solutions stabilize the PLA nanoparticles more effectively, and nanoparticles of less than 100 nm can be prepared. The cross-linked PMBT coating on the PLA nanoparticle surfaces can remarkably prevent the aggregation and phagocytosis of the coated particles. Furthermore, the antineoplastic drug DOX release is well controlled from the nanoparticles coated with PMBT. This simple and effective zwitterionic polymer-coating strategy may serve as a new route to design and optimize longcirculating controlled release nanoparticle drug carriers. Funding This work was supported by the National Natural Science Foundation of China [grant number 21244001], [grant number 21374087]; Japan Science Society [grant number S11-001] and the Fund of Engineering Laboratory of Xi’an City.
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