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Spatiotemporally Programmable Surface Engineered Nanoparticles for Effective Anticancer Drug Delivery Arsalan Ahmed, Hongliang Yu, Dingwang Han, Jingwei Rao, Yin Ding,* Yong Hu*
Surface engineered nanoparticles (NPs) are fabricated from polycaprolactone-polyethylenimine-folic acid (PCL-PEI-FA) and polycaprolactone-S-S-polyethylene glycol (PCL–S-S-PEG) copolymers. FESEM reveals the core-shell structure of these NPs of about 230 nm size. It is assumed that the inner cores of these NPs are composed of PCL, while the outer shells are adorned with PEG and folic acid, introducing a stealthy nature and specific targeting capability. Moreover, the disulfide bonds in the PCL–S-S-PEG copolymers provide a reduction-induced degradation characteristic in these NPs. Cell line experiments demonstrate the enhanced endocytosis and cytotoxicity of these NPs. Thus PCL-PEIFA/PCL-S-S-PEG NPs could be a better candidate for the tumor specific delivery of hydrophobic drugs.
Polymeric nanoparticles (NPs) can protect the load of drugs, shield the drug from a variety of in vivo degradation and digestive enzymes, control the expected rate of drug release, accumulate in tumor tissue, and increase the drug A. Ahmed, H. Yu, Prof. Y. Hu Institute of Materials Engineering, National Laboratory of Solid State Microstructure, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu 210093, P. R. China E-mail: [email protected] A. Ahmed, Y. Ding State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, P. R. China E-mail: [email protected] D. Han, J. Rao Hainan WeiKang Pharmaceutical (Qianshan) Co. Ltd., Anqing 246300, P. R. China
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
concentration at tumor sites due to the enhanced penetration effect.[1] The key factors required for a good drug carrier include long circulation time, tumor sensitivity, a specific binding ability to the cell membrane, and escape lysosomal membrane transport.[2] Although great research is being conducted in the field of drug loaded polymeric NPs, the real clinical use of these NPs is limited. Their main limitations are the short in vivo residence time due to the reticuloendothelial system (RES) and non-specific distribution of NPs in both tumor cells and normal cells.[3] Thus, for NPs, an integrated long circulation and specific targeting of tumor cells are great challenges.[4] Surface physics and the chemical effects of drug loaded NPs can be altered by means of surface modification, which enables these NPs to transport drug to tumor sites and regulate the drug release to achieve the best results.[5] Hydrophilic polymers, such as PEG, are coated on these NPs to avoid capture by the RES, enhance their in vivo circulation time, and thereby effectively improve the drug
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1. Introduction
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concentration at the tumor site.[6] Although these stealthy NPs reduce the non-specific cellular interactions, they also decrease the interaction between NPs and tumor cells, thus lessening the effectiveness of the anticancer drug in tumor cells.[7] In order to improve the impact of NPs on tumor cells, smart targeting polymeric NPs have been developed, i.e., pH targeting and tumor signaling molecule targeting NPs.[8] In these targeting technologies, the NP surface is modified by antibodies or ligands. These ligands bind to surface receptors on tumor cells, improve the interaction between NP and tumor cells and improve the drug delivery efficiency.[9–11] However, these modified targeting ligands on the NPs show tumor specific binding capacity only when in direct contact and do not show an appreciable tumor specific binding capacity during circulation in the body.[12,13] This is mainly because when NPs are circulating in the body, the surface ligands of NPs may interact with blood proteins or enzymes, blocking the effects of the ligands and diminishing the targeting role of the NPs on tumor cells. Therefore, whenever targeting ligands are directly exposed to body fluids, they cause no difference in the distribution of NPs between normal tissue and tumor tissue.[14] Thus, it is desirable to fabricate specific targeting NPs with a protective layer. This layer safeguards NPs against the body’s proteins and enzymatic attack, and maintains their long circulation characteristics. When NPs arrive at the tumor site, the protective layer is released either outside or inside the tumor cells (which is dependent upon the tumor signals, such as pH, temperature, or reducing environment), and the ligands bind to tumor cells due to the over expression of receptor. Recently, several types of multifunctional hybrid polymeric NPs with different structures and properties have been constructed by adjusting the type and proportion of copolymers.[15–17] Encouraged by this strategy, we have utilized step-by-step conjugation techniques to prepare two types of copolymers. One of the copolymers was obtained by binding PCL and targeting ligand FA. Since PCL and FA possess a similar reactive functional group, i.e., carboxylic acid groups, it is not possible to conjugate these two molecules directly. Therefore, first the terminal carboxylate groups of PCL were modified with the primary amine groups of PEI and then FA was reacted with the terminal amine group of PCL. Another block copolymer was obtained by binding PCL and PEG through the disulfide bond. This copolymer could introduce a stealthy nature and long circulating characteristics into NPs due to the PEG constituent. Furthermore, the disulfide linkage is reduction responsive and may break due to the reduction of the disulfide bond in the presence of a reducing agent, e.g., DTT or glutathione (GSH). These two copolymers were mixed in a certain proportion to obtain sandwich-structured polymeric NPs (Scheme 1). The outermost layer is a long
Scheme 1. Schematic diagram of PCL-PEI-FA/PCL-S-S-PEG NPs.
circulating layer consisting of PEG, the middle layer is the specific targeting layer of FA, and the innermost layer is the hydrophobic core of PCL in which to load a drug. The long circulating layer reduces the interaction of the NPs with proteins and other substances in body fluids, ensuring that the NPs are not macrophaged or recognized by the reticuloendothelial system during in vivo circulation, and further protects the middle targeting layer from interacting with other biological macromolecules. This long circulating layer will be eliminated from the surface of NPs after reaching the tumor site due to breakage of disulfide bonds by the reducing environment of tumor cells, and the targeting layer is then exposed.[18,19] The targeting layer enhances the interaction between NPs and tumor cells, increases the accumulation of NPs at the tumor site and facilitates tumor cells to engulf NPs. The innermost layer preserves the drug in its effective form, protects the drug from extracellular fluids and thus strengthens the toxicity of the drug against cancer cells.
2. Experimental Section 2.1. Materials PCL (M n , 10 kDa), methoxypolyethylene glycol thiol (PEG-thiol, 2 kDa), N-phenyltriazolinedione (PTAD), N-hydroxysuccinimide (NHS), cysteamine, and N,N‘dicyclohexyl carbodiimide (DCC) were purchased from Sigma-Aldrich and used as received. Polyethylenimine (PEI) with M n of 423 Da was purchased from Aladdin Chemical Co. Ltd and was purified by drying under reduced pressure. Folic acid (FA) was purchased from Beijing Aoboxing Biotech Co. Ltd. All other chemicals were of analytical grade.
2.2. Synthesis of PCL-PEI-FA PCL-PEI-FA was synthesized in two steps: the synthesis of PCL-PEI copolymer and then reaction with FA to obtain PCL-PEI-FA. Briefly, PCL (0.37 103 M), DCC (1.2 103 M), and NHS (1.2 103 M) solutions were prepared in THF, and mixed under magnetic stirring for 4 h at room temperature to obtain end group activated PCL. Later, a PEI solution (4.0 103 M) was added under magnetic stirring for 1 h. The insoluble byproduct was removed by filtration using 0.45 mm Teflon filter paper. The polymer product was
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precipitated into diethyl ether under vigorous stirring and was dissolved in methanol. The obtained polymer product was dialyzed against excess de-ionized water at 4 8C. The sample was then freezedried. Similarly, FA was activated by reacting FA (0.37 103 M) with DCC (1.2 103 M) and NHS (1.2 103 M) under magnetic stirring at room temperature for 4 h. PCL-PEI solution was added into it and magnetically stirred at room temperature for 1 h. The polymer product was then filtered, precipitated into diethyl ether and dissolved in methanol. Finally, the sample was freeze-dried and stored at –20 8C.
2.3. Synthesis of PCL-S-S-PEG PCL-S-S-PEG copolymer was prepared by the oxidation reaction of thiols to get disulfide copolymer using cysteamine and PEG-thiol, followed by conjugation of disulfide diblock polymer with activated PCL. Briefly, PEG-thiol (0.4 103 M), cysteamine (0.4 103 M), and PTAD (1.6 103 M) solutions were prepared in dry toluene, mixed together, and shaken gently. After decolorization, this mixture was washed with 10% NaOH twice and then with 25% NaCl solution. The organic phase was separated and dried over Na2SO4. The obtained disulfide copolymer was recrystallized from n-hexane/ethyl acetate mixture. The end carboxylic acid groups of PCL were activated by reaction with DCC and NHS under magnetic stirring for 4 h. The disulfide copolymer was mixed in it and stirred magnetically for 1 h. The insoluble byproduct was removed by filtration. Polymer product was precipitated in excess diethyl ether and dissolved in methanol. The polymer product was dialyzed against excess de-ionized water, freeze-dried, and stored at 20 8C.
2.4. Fabrication of PCL-PEI-FA/PCL-S-S-PEG NPs The desired amounts of two kinds of copolymers with different ratios were dissolved together in 1 mL of THF. Then, the copolymer solution was added dropwise into 10 mL of deionized water under gentle stirring at room temperature. Later, the solution was dialyzed extensively to remove the organic solution. Finally, the resulting solution was filtered through a 600 nm filter membrane to remove copolymer aggregates. The obtained nanoparticles were abbreviated as PCL-PEI-FA/PCL-S-S-PEG NPs.
2.5. Drug Encapsulation Efficiency The PTX content in PCL-PEI-FA/PCL-S-S-PEG NPs was determined by HPLC (Waters 2695, Milford, MA). 5 mg of PCL-PEI-FA/PCL-S-S-PEG NPs were dissolved in 1 mL of dichloromethane (DCM) by vigorous shaking and then 5 mL of a mixture of methanol, water, and acetonitrile in the ratio 23:41:36 was added to it. DCM was removed completely by purging with N2 until a clear solution was obtained. After filtration, the resulting solution was analyzed using HPLC analysis. The mobile phase consisted of methanol, water, and acetonitrile in the ratio 23:41:36. The column effluent was detected at 242 nm with a UV-vis detector (Waters 2487, dual l absorbance detector). The measurement was performed thrice. The drug encapsulation efficiency (DEE) was denoted as the percentage
DEE ¼
Actual amount of drug in NPs 100 Feeding amount of drug in NPs
ð1Þ
2.6. In Vitro Drug Release Experiment The in vitro release of PTX from the PCL-PEI-FA/PCL-S-S-PEG NPs was investigated by dialyzing a NP suspension in 50 103 M PBS with or without 10 103 M DTT at 37 8C. Briefly, 1 mL of NP suspension was put in a dialysis tube (MWCO 12 000) and dialyzed against both types of media, i.e., PBS and PBS þ DTT media. After certain time intervals, 6 mL of release media were taken out and refilled with an equal volume of fresh medium. 1 mL of DCM was added to the release medium, shaken well, and separated the organic layer. Later, the organic layer was mixed with 5 mL of a mixture of methanol, water, and acetonitrile, and DCM was evaporated completely by purging with N2 gas. The released amount of PTX in the resulting solution was determined by HPLC. The release experiments were conducted in triplicate and the results presented here are the average data.
2.7. Cell Viability Assay MCF-7 breast cancer cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin at 37 8C in a humidified environment of 5% CO2. Before performing the experiments, the cells were pre-cultured until confluence was reached. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to study the cytotoxicity of PCL-PEI-FA/PCL-S-S-PEG NPs against MCF-7 breast cancer cells. The cells were seeded in 96-well plates at 4 104 cells ml1. After 24 h incubation, the medium was replenished with PCLPEI-FA/PCL-S-S-PEG NP suspensions of different concentrations. The cells were incubated for 24, 48, and 72 h, respectively. After this, wells were washed thrice with PBS. 10 mL of freshly prepared MTT solution and 90 mL of medium were added to each well. After incubating for approximately another 4 h, MTT medium solution was removed leaving a precipitate. 100 mL of DMSO were added to each well and the plate was shaken gently in order to dissolve the precipitate. The absorbance of MTT at 570 nm was observed using a microplate reader after subtracting the absorbance of the corresponding control wells. Cell viability was represented as the ratio of the absorbance of the cells incubated with NP suspension to that of the cells incubated with culture medium only (Equation (2)). Cell Viability ¼
Absorbance of cells treated with drug loaded NPs Absorbance of untreated cells 100
ð2Þ
2.8. Cellular Uptake MCF-7 breast cancer cells were cultured in the confocal imaging chambers (LAB-TEK, Chambered Coverglass System) at 37 8C. The
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of the drug loaded in the final product (Equation (1)).
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medium was changed after an appropriate interval until 80% confluence was reached. The medium was removed and cells were washed thrice with PBS. Coumarin-6 loaded NPs were then placed into the confocal imaging chamber, and cells were incubated for 8 h. Later, cells were washed with PBS thrice, fixed with 75% ethanol for 20 min and further washed with PBS twice. Finally, the cells were imaged using a Confocal Laser Scanning Microscope (CLSM, Zeiss LSM 410, Jena, Germany) with imaging software (Fluoview FV1000).
2.9. Characterization FTIR was carried out using a Bruker IFS 66 V vacuum-type spectrometer in a range between 4000 and 500 cm1 by mixing freeze-dried copolymer powder with KBr. 1H NMR was carried out using a Bruker MSL-300 spectrometer with tetramethylsilane as an internal standard. GPC was conducted utilizing a Waters 244 gel permeation chromatograph instrument with tetrahydrofuran as the eluent at a flow rate of 1 mL min1. UV-vis spectroscopy was used to obtain the folic acid content of PCL-PEI-FA on the basis of a standard curve. PCL-PEI-FA polymer solution and FA solutions of different concentrations were prepared. The absorbance of these solutions was measured from 200 to 500 nm with a Shimadzu UV-3600 spectrophotometer and the FA content was determined at 280 nm. The particle sizes of PCL-PEI-FA/PCL-S-S-PEG NPs were determined by the dynamic laser scattering (DLS) technique (Brookhaven Instruments Corporation 90 plus Particle Sizer, USA) at 25 8C with at a scattering angle of 908. The zeta potential of the NPs was measured using laser Doppler anemometry (Zeta Plus, Zeta Potential Analyzer, Brookhaven Instruments Corporation, USA). Prior to zeta potential calculations, the NP suspension was diluted in de-ionized water. The value was recorded as the average of three measurements. The morphology of the NPs was observed by scanning electron microscopy (FESEM, JEOL JSM- 6700F, Japan) and transmission electron microscopy (TEM, JEOL TEM-100, Japan). The size change in the micelles, due to destabilization and agglomeration, in response to DTT in PBS was monitored using DLS measurements. Briefly, 10 103 M DTT solution was obtained by mixing 1.5 mL of PCL-PEI-FA/PCL-S-S-PEG NP suspension and DTT in 50 103 M PBS. It was then placed in a shaking bath with a speed of 200 rpm at 37 8C. The micelle size was measured at appropriate intervals using the DLS technique.
3. Results and Discussion 3.1. Synthesis and Analysis of Copolymers A conjugation technique was utilized to synthesize PCL-PEIFA and PCL-S-S-PEG block copolymers. The carboxylic acid group of PCL can be coupled to the primary amine of PEI using DCC and NHS conjugation agents.[20] In brief, the reaction of the carboxylic acid of PCL with NHS and DCC produces DCC activated ester. These activated esters can be coupled with PEI to form stable conjugated PCL-PEI copolymer.[21] Since PEI has amine groups attached at both
ends, an excess amount of PEI was used in order to prohibit the synthesis of PCL-PEI-PCL.[22] Similarly, FA was activated via DCC and NHS and thereafter conjugated with PCL-PEI to produce PCL-PEI-FA copolymer, as shown in Scheme 2. Disulfide containing copolymer was synthesized by the oxidation reaction of thiols to yield disulfide containing PEG, and it was then conjugated with PCL. PTAD is considered to be an efficient reagent for the oxidation of thiols to their respective disulfide compound.[23] Herein PEG-SH was reacted with cysteamine using PTAD to obtain PCL-cysteamine, and later PCL-cysteamine was coupled with activated PCL to produce PCL-S-S-PEG (Scheme 2b). Figure 1 shows the FTIR spectra of PCL, PCL-PEI-FA, PEGthiol, and PCL-S-S-PEG. PCL shows the representative peaks of carbonyl groups at 1720 cm1, C—H peaks of aliphatic groups at 2947 cm1 and 2865 cm1, and carbon-oxygen stretching at 1170 cm1.[24] In the spectrum of PCL-PEI-FA, the characteristic peaks of amine at 3321 cm1 and 1550 cm1, and the carbonyl group peak of amide at 1650 cm1 describe the successful coupling of PCL with PEI.[25] The representative peaks of PEG at 2850–3000 cm1 for CH2 groups, 1380 cm1 for C—H bending, and 1100 cm1 for C—O—C stretching, along with the peaks of the SH group at 2505 cm1 were obtained in the PEG-thiol spectrum.[26] However, the SH group peak vanishes and a new peak for CH2—S originates at 1270 cm1 in PCL-S-S-PEG copolymer.[27] The representative peaks of PCL and the carbonyl peak of amide at 1650 cm1 were also observed in the PCL-S-S-PEG spectrum, which clearly indicates the formation of PCL-S-S-PEG. The chemical structures of these synthesized copolymers were also verified by 1H NMR, as shown in Figure 2. Figure 2a shows the 1H NMR spectrum of PCL-PEI-FA, which demonstrates the four major characteristic peaks of PCL at 1.4 (c), 1.6 (b), 2.3 (a), and 4.1 (d) ppm, attributed to the repeating units of PCL.[28] The peak at about 2.7 (e) ppm is related to PEI,[29] and the characteristic peaks of FA at 6.5– 7.0 (f) ppm can be clearly observed. PCL-S-S-PEG also showed characteristic peaks of PEG at about 3.7 and 3.4 ppm, and the peaks of disulfide linkages at about 2.85 and 2.92 ppm (Figure 2b).[30] Based on this data from 1H NMR, together with the results from FT-IR, it seems safe to claim that PCL-PEI-FA and PCL-S-S-PEG were successfully obtained using the conjugation technology. The content of FA in the PCL-FEI-FA has a great effect on its targeting ability to the tumor. Thus, the crosslinking of FA with PCL-PEI and its content were investigated, with the UV-vis spectrum of PCL-PEI-FA shown in Figure 3. The characteristic absorbance peak of FA at 360 nm was observed both in PCL-PEI-FA and the standards, which confirmed the successful conjugation of FA onto PCL-PEI copolymer.[31] Later, the FA content in the PCL-PEI-FA was determined by the standard curve method and was about 0.019 mg mL1. This value indicated that 96% (mol) of FA
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Scheme 2. Chemical pathways for copolymer synthesis.
was conjugated onto PCL-PEI, calculated from the feeding amount. The content of FA in the polymer could also be controlled through changing the feeding amount. The molecular weight of these obtained copolymers was measured with GPC, which is shown in Figure 4. After
the conjugation, narrow molecular weight distributions of copolymers with M n of 14 100 Da and 12 200 Da for PCL-S-SPEG and PCL-PEI-FA were observed, respectively. Considering the molecular weight of the original polymer (PCL, 10 000 Da, PEG-SH, 2000 Da), we thought that PCL-S-S-PEG
Figure 1. FTIR spectra of (a) PCL, (b) PCL-PEI-FA, (c) PEG-thiol and (d) PCL-S-S-PEG.
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Figure 2. 1H NMR spectra of (a) PCL-PEI-FA and (b) PCL-S-S-PEG.
Figure 3. UV-vis spectra of (a) PCL-PEI-FA with standard concentrations of FA and (b) their standard curve.
was successfully obtained. The polydispersity index (PDI) was 1.57 for PCL-S-S-PEG and 1.32 for PCL-PEI-FA. The single peaks of copolymers, as shown in Figure 4, show the monodispersity of the molecular weights of the copolymers and the absence of PCL-PEI-PCL formation during the conjugation reaction. 3.2. The Size and Morphology of PCL-PEI-FA/PCL-S-SPEG NPs Copolymers with both hydrophobic and hydrophilic parts normally assemble into core-shell type NPs in aqueous solution due to their amphiphilic nature.[32] Similarly, in
this work, core-shell nanoparticles were obtained by mixing two kinds of amphiphilic copolymers, i.e., PCLPEI-FA and PCL-S-S-PEG. The surface morphology of PCLPEI-FA/PCL-S-S-PEG NPs was characterized with FESEM, as shown in Figure 5a. The shape and size of these NPs was investigated by TEM (Figure 5b). PCL-PEI-FA/PCL-S-S-PEG NPs showed a spherical shape with a uniform size of approximately 230 nm. Their size and size distribution were further investigated using DLS. Figure 5b shows DLS data for PCL-PEI-FA/PCL-S-S-PEG NPs in aqueous medium. It was observed that the NPs exhibited a unimodal size distribution and the average NP size obtained from DLS data was about 270 nm. This size is a little bigger than
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Figure 4. GPC chromatograms of copolymers.
that of FESEM, because DLS gives the hydrodynamic mean diameter while FESEM provides the mean diameter in the dry state. Figure 6a describes the zeta potential trend of PCL-PEI-FA/ PCL-S-S-PEG NPs with an increasing proportion of PCL-PEIFA copolymer. When the mole ratio of PCL-PEI-FA in the NPs increased from 5% to 20%, the zeta potential of PCL-PEI-FA/ PCL-S-S-PEG NPs increased from 2.2 mV to 7.1 mV. This change in zeta potential is reasonable because PCL-PEI-FA is a positively charged polymer due to the PEI segment. Thus, increasing the ratio of PCL-PEI-FA copolymer causes an increment in the zeta potential of these NPs. Positive zeta potential is an essential requirement for the endocytosis of NPs by tumor cells due to the electrostatic attraction between positively charged NPs and negatively charged cell membranes.[33] Based on the above fact, the ratio of PCL-PEI-FA could be adjusted to be as high as possible in order to get effective binding ability.
Figure 5. SEM image of PCL-S-S-PEG/PCL-PEI-FA NPs with their surface morphology (inset) (a); TEM image of NPs (b); DLS result for NPs in aqueous solution (c).
Figure 6. Zeta potential of NPs (a) and size of NPs (b) with different proportions of PCL-PEI-FA to PCL-S-S-PEG.
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The size of PCL-PEI-FA/PCL-S-S-PEG NPs with different ratios of PCL-PEI-FA to PCL-S-S-PEG was measured by DLS and is shown in Figure 6b. It can be clearly observed that PCL-PEI-FA/PCL-S-S-PEG NPs enlarged when the amount of PCL-S-S-PEG copolymer was increased. Since PCL-PEI-FA/ PCL-S-S-PEG NPs are composed of a core-shell structure, where PCL is the core and PEG is the shell, increments in the proportion of PCL-S-S-PEG copolymers in the NPs result in thickening of the outermost PEG layer. As a result, the size of NPs increases. 3.3. Reduction Triggered Destabilization Cancer cells possess an internal reductive environment due to a high level of presence of antioxidant-Glutathione.[34] In order to obtain an in vitro reductive environment similar to cancer cells, DTT is commonly used to obtain reductioninduced degradation. The disulfide bond is cleavable in the presence of a reducing agent such as DTT. Herein, DTT reduces the disulfide linkages of PCL-S-S-PEG to thiols via two sequential thiol-disulfide exchange reactions.[35] After the cleavage of the disulfide bond of PCL-S-S-PEG, PCL-SH and PEG-SH are formed. The PEG-SH would detach from the PCL-PEI-FA/PCL-S-S-PEG NPs, exposing targeting ligands. This overall phenomenon will cause destabilization and the aggregation of NPs, which will improve the NPs’ targeting ability to tumor cells and stimulate the uptake of the NPs by tumor cells. To test this point, 10 103 M DTT in 50 103 M PBS at pH 7.4 was used to mimic the reductive physiological environment and facilitate the reduction-induced destabilization of the disulfide bonds of PCL-S-S-PEG. The size change of PCLPEI-FA/PCL-S-S-PEG NPs in response to DTT was investigated by DLS, as shown in Figure 7. The average particle size of PCL-PEI-FA/PCL-S-S-PEG NPs remained constant in the absence of DTT during the testing period. However, after the addition of DTT, the size of PCL-PEI-FA/PCL-S-SPEG NPs increased from 270 nm to about 1000 nm within 60 min, which clearly confirmed that the PEG segments were detached from the nanoparticles, resulting in particle aggregation and break up of the NPs. Moreover, this destabilization of the NPs was enhanced with an increase in the proportion of PCL-S-S-PEG copolymer in the NPs, since a higher amount of PCL-S-SPEG was conducive to a higher detachment rate of PEG and more serious particle aggregation. 3.4. Drug Encapsulation and Drug Release Behavior of NPs Core-shell NPs with a hydrophobic interior and a hydrophilic exterior environment are promising candidates for encapsulating hydrophobic drug in their cores, while the hydrophilic shell stabilizes NPs in aqueous media.[36]
Figure 7. Size change of PCL-PEI-FA/PCL-S-S-PEG micelles in response to DTT measured by DLS.
Herein, PTX was selected as a model drug to encapsulate inside these PCL-PEI-FA/PCL-S-S-PEG NPs. The drug loading ability and in vitro release profiles are depicted in Figure 8. Figure 8a describes the PTX encapsulation efficiency of NPs against the PCL-PET-FA mole ratio inside the NPs. It was found that as the ratio of PCL-PEI-FA increased, the PTX encapsulation efficiency decreased. We thought PEG, being a hydrophilic shell, played a vital role in the stabilization of the NPs. A high proportion of PCL-S-S-PEG copolymer in the NPs enhanced the stability of PCL-PEI-FA/PCL-S-S-PEG NPs, and gave high yields of NPs. After increasing the ratio of PCL-PEI-FA, the stability of PCL-PEI-FA/PCL-S-S-PEG NPs decreased; more PTX loaded NPs would congregate and form large aggregates, which would be filtered out during the preparation procedure. Thus lower drug loading efficiency was obtained. The drug release profile of PTX loaded NPs with and without DTT is shown in Figure 8b. For all samples with different polymer ratios, less than 8% of encapsulated PTX was released from the PCL-PEI-FA/PCL-S-S-PEG NPs within 20 h, which indicated that PCL-PEI-FA/PCL-S-S-PEG NPs encapsulated PTX very well and provided a sustained drug release profile. However, in the presence of DTT, an abrupt release of PTX was found and up to 82% of PTX was released out from the NPs in 20 h. Moreover, the PTX released from NPs with a high proportion of PCL-S-S-PEG was greater when compared to those with a low proportion. The split of the disulfide bonds causes the detachment of PEG and the structural breakdown of the NPs, which consequently enables a quick release of PTX. As the proportion of PCL-S-S-PEG increased, more PCL-S-S-PEG involved inside the NPs would break up due to DTT.
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Figure 8. PTX encapsulation efficiency of NPs with different ratios of copolymers (a) and PTX release behavior of NPs with and without DTT (b).
Therefore, the break up rate of the NPs is accelerated, which enables a faster drug release. 3.5. Cell Viability and Cellular Uptake of NPs The cytotoxicity of drug loaded NPs is greatly related to the binding and cellular uptake capabilities of the NPs to the targeting cells. In this work, the PEG chain of PCL-PEI-FA/ PCL-S-S-PEG NPs shields the positively charged PEI segment, and also protects the FA ligand from contact with the biomacromolecules before the NPs reach the targeting site. At the tumor site, the PEG layer detaches from the NPs, and PEI segments and FA ligands are exposed to the tumor cells directly. The positively charged PEI segment will enhance the binding ability of NPs with tumor cells due to the static electronic interaction.[37] The presence of FA will boost the tumor cells’ selective uptake of these NPs because there are many FA receptors on the surface of tumor cells. In order to evaluate the cytotoxicity of PCL-PEI-FA/PCL-S-S-PEG NPs, the viability of the MCF-7 cell line was tested in the presence of empty and drug loaded NPs. Figure 9 describes the cytotoxicity of empty and PTX loaded NPs incubated with MCF-7 cells for 24, 48, and 72 h using the MTT assay. It apparently showed that empty NPs had very little toxicity against MCF-7 cells, whereas PTX loaded NPs exhibited noticeable cytotoxicity with increasing PTX concentration. Moreover, the toxicity of drug loaded NPs increased with the passage of time. This could be attributed to the sustained release of PTX from NPs. After incubation for 72 h, the highest cytotoxicity of PTX loaded NPs was observed. This implies that the amount of the PTX released
Figure 9. Cell viability of MCF-7 cells incubated with blank and PTX loaded NPs.
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from the NPs is high enough to suppress the proliferation of MCF-7 cells. It has been reported that stealthy NPs with specific targeting characteristics can escape from endosomes and be delivered to cytoplasmic organelles.[38] The fate and the cellular uptake of Coumarin-6 loaded PCL-PEI/PCL-S-S-PEG NPs and Coumarin-6 loaded PCL-PEI-FA/PCL-S-S-PEG NPs was studied by CLSM incubated with the MCF-7 cell line for 6 h. Coumarin-6 showed green fluorescence in intracellular compartments such as cytosol and cell nucleus after 6 h
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Figure 10. CLSM of MCF-7 cells incubated for 6 h with coumarin-6 loaded PCL-PEI/ PCL-S-S-PEG NPs (a) and PCL-PEI-FA/PCL-S-S-PEG NPs (b).
incubation with both types of NPs, which indicated the rapid engulfment of NPs and the release of Coumarin-6 inside cells (Figure 10). The disulfide bond is broken in the intracellular compartments because of the relatively high concentration of reducing glutathione tripeptide,[39] which could accelerate intracellular distribution of coumarin-6.
4. Conclusion In order to fabricate multifunctional NPs for cancer therapy, two types of copolymer were synthesized via conjugation techniques; PCL-PEI-FA contains specific targeting characteristics while PCL-S-S-PEG possesses a long circulation property. Furthermore, the disulfide linkage of PCL-S-S-PEG was utilized to generate reduction-induced destabilization capabilities in these NPs. By adjusting the ratio of these copolymers, core-shell structured NPs of approximately 230 nm were obtained. These core-shell structured NPs showed enhanced stability and PTX encapsulation efficiency. However, these NPs exhibited quick PTX release in a reductive environment, which improved their effectiveness against tumors. Moreover, the long circulation and
specific targeting qualities of PEG and FA embedded in the outer layer could make this system an efficient delivery platform for anticancer drugs.
Acknowledgements: This work was supported by the National Natural Science Foundation of China (No. 21105047 and 51173077), and Fundamental Research Funds for the Central Universities (1085021309).
Received: May 8, 2014; Revised: July 12, 2014; Published online: DOI: 10.1002/mabi.201400228 Keywords: core-shell structures; long circulation; nanoparticles, polycaprolactone; reduction induced degradation
[1] L. Zhang, Y. Hu, X. Jiang, C. Yang, Z. W. Lu, Y. H. Yang, J. Controlled Release 2004, 96, 135. [2] V. J. Venditto, F. C. Szoka, Adv. Drug Delivery Rev. 2013, 65, 80. [3] G. S. Kwon, T. Okano, Adv. Drug Delivery Rev. 1996, 21, 107. [4] R. Weissleder, K. Kelly, E. Y. Sun, T. Shtatland, L. Josephson, Nat. Biotechnol. 2005, 23, 1418. [5] M. L. Hans, A. M. Lowman, Curr. Opin. Solid St. M. 2002, 6, 319.
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[6] R. Gref, Y. Minamitake, M. T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer, Science 1994, 263, 1600. [7] Y. Hu, J. Xie, Y. W. Tong, C. H. Wang, J. Controlled Release 2007, 118, 7. [8] Brigger, C. Dubernet, P. Couvreur, Adv. Drug Delivery Rev. 2002, 54, 631. [9] C. P. Leamon, J. A. Reddy, Adv. Drug Delivery Rev. 2004, 56, 1127. [10] H. S. Yoo, T. G. Park, J. Controlled Release 2004, 96, 273. [11] Z. M. Qian, H. Li, H. Sun, K. Ho, Pharmacol. Rev. 2002, 54, 561. [12] C.-H. J. Choi, C. A. Alabi, P. Webster, M. E. Davis, Proc. Natl. Acad. Sci. USA 2010, 107(3), 1235. [13] D. B. Kirpotin, D. C. Drummond, Y. Shao, M. R. Shalaby, K. Hong, U. B. Nielsen, J. D. Marks, C. C. Benz, J. W. Park, Cancer Res. 2006, 66, 6732. [14] T. M. Allen, Nat. Rev. Cancer 2002, 2, 750. [15] G. Li, L. Shi, R. Ma, Y. An, N. Huang, Angew. Chem. Int. Ed. 2006, 45, 4959. [16] C. L. Lo, C. K. Huang, K. M. Lin, G. H. Hsiue, Biomaterials 2007, 28, 1225. [17] F. Gu, L. F. Zhang, B. A. Teply, N. Mann, A. Wang, A. F. RadovicMoreno, R. Langer, O. C. Farokhzad, Proc. Natl. Acad. Sci. USA 2008, 105(7), 2586. [18] E. P. Feener, W. C. Shen, H. J.-P. Ryser, J. Biol. Chem. 1990, 26(31), 18780. [19] W. C. Sun, P. B. Davis, J. Controlled Release 2010, 146, 118. [20] J. J. Yoon, Y. S. Nam, J. H. Kim, T. G. Park, Biotechnol. Bioeng. 2002, 78, 1. [21] G. T. Hermanson, Bioconjugate Technique, 2nd ed., Elsevier, London 2008, p. 139. [22] Y. S. Nam, H. S. Kang, J. Y. Park, T. G. Park, S. H. Han, I. S. Chang, Biomaterials 2003, 24, 2053.
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[23] A. Christoforou, G. Nicolaou, Y. Elemes, Tetrahedron Lett. 2006, 47, 9211. [24] F. Causa, E. Battista, R. D. Moglie, D. Guarnieri, M. Iannone, P. A. Netti, Langmuir 2010, 26, 9875. [25] S. Yuan, G. Xiong, X. Wang, S. Zhang, C. Choong, J. Mater. Chem. 2012, 22, 13039. [26] J. Manson, D. Kumar, B. J. Meenan, D. Dixon, Gold Bull. 2011, 44, 99. [27] W. H. Yang, W. W. Li, H. J. Dou, K. Sun, Mater. Lett. 2008, 62, 2564. [28] S. Aryal, R. Bahadur, N. Bhattarai, B. M. Lee, H. Y. Kim, Mater. Chem. Phys. 2006, 98, 463. [29] C. H. Wang, G. H. Hsiue, Bioconjugate Chem. 2005, 16, 391. [30] W. Wang, H. Sun, F. Meng, S. Ma, H. Liu, Z. Zhong, Soft Matter 2012, 8, 3949. [31] L. Liu, M. Zheng, T. Renette, T. Kissel, Bioconjugate Chem. 2012, 23, 1211. [32] Y. S. Siu, L. Li, M. F. Leung, K. L. Daniel Lee, P. Li, Biointerphases 2012, 7, 16. [33] H. Cheng, J. L. Zhu, X. Zeng, Y. Jing, X. Z. Zhang, R. X. Zhuo, Bioconjugate Chem. 2009, 20, 481. [34] G. K. Balendiran, R. Dabur, D. Fraser, Cell Biochem. Funct. 2004, 22, 343. [35] H. Y. Wen, H. Q. Dong, W. J. Xie, Y. Y. Li, K. Wang, G. M. Pauletti, D. L. Shi, Chem. Commun. 2011, 47, 3550. [36] T. Riley, S. Stolnik, C. R. Heald, C. D. Xiong, M. C. Garnett, L. Illum, S. S. Davis, Langmuir 2001, 17, 3168. [37] J. Yang, E. J. Cho, S. Seo, J. W. Lee, H. G. Yoon, J. S. Suh, Y. M. Huh, S. Haam, Biomed. Mater. Res. A 2008, 84, 273. [38] R. Savic, A. Eisenberg, D. Maysinger, J. Drug Target. 2006, 14, 343. [39] F. Meng, W. E. Hennink, Z. Zhong, Biomaterials 2009, 30, 2180.
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Spatiotemporally Programmable Surface Engineered Nanoparticles for Effective Anticancer Drug Delivery Arsalan Ahmed, Hongliang Yu, Dingwang Han, Jingwei Rao, Yin Ding,* Yong Hu*
Surface engineered nanoparticles (NPs) are fabricated from polycaprolactone-polyethylenimine-folic acid (PCL-PEI-FA) and polycaprolactone-S-S-polyethylene glycol (PCL–S-S-PEG) copolymers. FESEM reveals the core-shell structure of these NPs of about 230 nm size. It is assumed that the inner cores of these NPs are composed of PCL, while the outer shells are adorned with PEG and folic acid, introducing a stealthy nature and specific targeting capability. Moreover, the disulfide bonds in the PCL–S-S-PEG copolymers provide a reduction-induced degradation characteristic in these NPs. Cell line experiments demonstrate the enhanced endocytosis and cytotoxicity of these NPs. Thus PCL-PEIFA/PCL-S-S-PEG NPs could be a better candidate for the tumor specific delivery of hydrophobic drugs.
Polymeric nanoparticles (NPs) can protect the load of drugs, shield the drug from a variety of in vivo degradation and digestive enzymes, control the expected rate of drug release, accumulate in tumor tissue, and increase the drug A. Ahmed, H. Yu, Prof. Y. Hu Institute of Materials Engineering, National Laboratory of Solid State Microstructure, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu 210093, P. R. China E-mail: [email protected] A. Ahmed, Y. Ding State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu 210093, P. R. China E-mail: [email protected] D. Han, J. Rao Hainan WeiKang Pharmaceutical (Qianshan) Co. Ltd., Anqing 246300, P. R. China
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concentration at tumor sites due to the enhanced penetration effect.[1] The key factors required for a good drug carrier include long circulation time, tumor sensitivity, a specific binding ability to the cell membrane, and escape lysosomal membrane transport.[2] Although great research is being conducted in the field of drug loaded polymeric NPs, the real clinical use of these NPs is limited. Their main limitations are the short in vivo residence time due to the reticuloendothelial system (RES) and non-specific distribution of NPs in both tumor cells and normal cells.[3] Thus, for NPs, an integrated long circulation and specific targeting of tumor cells are great challenges.[4] Surface physics and the chemical effects of drug loaded NPs can be altered by means of surface modification, which enables these NPs to transport drug to tumor sites and regulate the drug release to achieve the best results.[5] Hydrophilic polymers, such as PEG, are coated on these NPs to avoid capture by the RES, enhance their in vivo circulation time, and thereby effectively improve the drug
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concentration at the tumor site.[6] Although these stealthy NPs reduce the non-specific cellular interactions, they also decrease the interaction between NPs and tumor cells, thus lessening the effectiveness of the anticancer drug in tumor cells.[7] In order to improve the impact of NPs on tumor cells, smart targeting polymeric NPs have been developed, i.e., pH targeting and tumor signaling molecule targeting NPs.[8] In these targeting technologies, the NP surface is modified by antibodies or ligands. These ligands bind to surface receptors on tumor cells, improve the interaction between NP and tumor cells and improve the drug delivery efficiency.[9–11] However, these modified targeting ligands on the NPs show tumor specific binding capacity only when in direct contact and do not show an appreciable tumor specific binding capacity during circulation in the body.[12,13] This is mainly because when NPs are circulating in the body, the surface ligands of NPs may interact with blood proteins or enzymes, blocking the effects of the ligands and diminishing the targeting role of the NPs on tumor cells. Therefore, whenever targeting ligands are directly exposed to body fluids, they cause no difference in the distribution of NPs between normal tissue and tumor tissue.[14] Thus, it is desirable to fabricate specific targeting NPs with a protective layer. This layer safeguards NPs against the body’s proteins and enzymatic attack, and maintains their long circulation characteristics. When NPs arrive at the tumor site, the protective layer is released either outside or inside the tumor cells (which is dependent upon the tumor signals, such as pH, temperature, or reducing environment), and the ligands bind to tumor cells due to the over expression of receptor. Recently, several types of multifunctional hybrid polymeric NPs with different structures and properties have been constructed by adjusting the type and proportion of copolymers.[15–17] Encouraged by this strategy, we have utilized step-by-step conjugation techniques to prepare two types of copolymers. One of the copolymers was obtained by binding PCL and targeting ligand FA. Since PCL and FA possess a similar reactive functional group, i.e., carboxylic acid groups, it is not possible to conjugate these two molecules directly. Therefore, first the terminal carboxylate groups of PCL were modified with the primary amine groups of PEI and then FA was reacted with the terminal amine group of PCL. Another block copolymer was obtained by binding PCL and PEG through the disulfide bond. This copolymer could introduce a stealthy nature and long circulating characteristics into NPs due to the PEG constituent. Furthermore, the disulfide linkage is reduction responsive and may break due to the reduction of the disulfide bond in the presence of a reducing agent, e.g., DTT or glutathione (GSH). These two copolymers were mixed in a certain proportion to obtain sandwich-structured polymeric NPs (Scheme 1). The outermost layer is a long
Scheme 1. Schematic diagram of PCL-PEI-FA/PCL-S-S-PEG NPs.
circulating layer consisting of PEG, the middle layer is the specific targeting layer of FA, and the innermost layer is the hydrophobic core of PCL in which to load a drug. The long circulating layer reduces the interaction of the NPs with proteins and other substances in body fluids, ensuring that the NPs are not macrophaged or recognized by the reticuloendothelial system during in vivo circulation, and further protects the middle targeting layer from interacting with other biological macromolecules. This long circulating layer will be eliminated from the surface of NPs after reaching the tumor site due to breakage of disulfide bonds by the reducing environment of tumor cells, and the targeting layer is then exposed.[18,19] The targeting layer enhances the interaction between NPs and tumor cells, increases the accumulation of NPs at the tumor site and facilitates tumor cells to engulf NPs. The innermost layer preserves the drug in its effective form, protects the drug from extracellular fluids and thus strengthens the toxicity of the drug against cancer cells.
2. Experimental Section 2.1. Materials PCL (M n , 10 kDa), methoxypolyethylene glycol thiol (PEG-thiol, 2 kDa), N-phenyltriazolinedione (PTAD), N-hydroxysuccinimide (NHS), cysteamine, and N,N‘dicyclohexyl carbodiimide (DCC) were purchased from Sigma-Aldrich and used as received. Polyethylenimine (PEI) with M n of 423 Da was purchased from Aladdin Chemical Co. Ltd and was purified by drying under reduced pressure. Folic acid (FA) was purchased from Beijing Aoboxing Biotech Co. Ltd. All other chemicals were of analytical grade.
2.2. Synthesis of PCL-PEI-FA PCL-PEI-FA was synthesized in two steps: the synthesis of PCL-PEI copolymer and then reaction with FA to obtain PCL-PEI-FA. Briefly, PCL (0.37 103 M), DCC (1.2 103 M), and NHS (1.2 103 M) solutions were prepared in THF, and mixed under magnetic stirring for 4 h at room temperature to obtain end group activated PCL. Later, a PEI solution (4.0 103 M) was added under magnetic stirring for 1 h. The insoluble byproduct was removed by filtration using 0.45 mm Teflon filter paper. The polymer product was
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precipitated into diethyl ether under vigorous stirring and was dissolved in methanol. The obtained polymer product was dialyzed against excess de-ionized water at 4 8C. The sample was then freezedried. Similarly, FA was activated by reacting FA (0.37 103 M) with DCC (1.2 103 M) and NHS (1.2 103 M) under magnetic stirring at room temperature for 4 h. PCL-PEI solution was added into it and magnetically stirred at room temperature for 1 h. The polymer product was then filtered, precipitated into diethyl ether and dissolved in methanol. Finally, the sample was freeze-dried and stored at –20 8C.
2.3. Synthesis of PCL-S-S-PEG PCL-S-S-PEG copolymer was prepared by the oxidation reaction of thiols to get disulfide copolymer using cysteamine and PEG-thiol, followed by conjugation of disulfide diblock polymer with activated PCL. Briefly, PEG-thiol (0.4 103 M), cysteamine (0.4 103 M), and PTAD (1.6 103 M) solutions were prepared in dry toluene, mixed together, and shaken gently. After decolorization, this mixture was washed with 10% NaOH twice and then with 25% NaCl solution. The organic phase was separated and dried over Na2SO4. The obtained disulfide copolymer was recrystallized from n-hexane/ethyl acetate mixture. The end carboxylic acid groups of PCL were activated by reaction with DCC and NHS under magnetic stirring for 4 h. The disulfide copolymer was mixed in it and stirred magnetically for 1 h. The insoluble byproduct was removed by filtration. Polymer product was precipitated in excess diethyl ether and dissolved in methanol. The polymer product was dialyzed against excess de-ionized water, freeze-dried, and stored at 20 8C.
2.4. Fabrication of PCL-PEI-FA/PCL-S-S-PEG NPs The desired amounts of two kinds of copolymers with different ratios were dissolved together in 1 mL of THF. Then, the copolymer solution was added dropwise into 10 mL of deionized water under gentle stirring at room temperature. Later, the solution was dialyzed extensively to remove the organic solution. Finally, the resulting solution was filtered through a 600 nm filter membrane to remove copolymer aggregates. The obtained nanoparticles were abbreviated as PCL-PEI-FA/PCL-S-S-PEG NPs.
2.5. Drug Encapsulation Efficiency The PTX content in PCL-PEI-FA/PCL-S-S-PEG NPs was determined by HPLC (Waters 2695, Milford, MA). 5 mg of PCL-PEI-FA/PCL-S-S-PEG NPs were dissolved in 1 mL of dichloromethane (DCM) by vigorous shaking and then 5 mL of a mixture of methanol, water, and acetonitrile in the ratio 23:41:36 was added to it. DCM was removed completely by purging with N2 until a clear solution was obtained. After filtration, the resulting solution was analyzed using HPLC analysis. The mobile phase consisted of methanol, water, and acetonitrile in the ratio 23:41:36. The column effluent was detected at 242 nm with a UV-vis detector (Waters 2487, dual l absorbance detector). The measurement was performed thrice. The drug encapsulation efficiency (DEE) was denoted as the percentage
DEE ¼
Actual amount of drug in NPs 100 Feeding amount of drug in NPs
ð1Þ
2.6. In Vitro Drug Release Experiment The in vitro release of PTX from the PCL-PEI-FA/PCL-S-S-PEG NPs was investigated by dialyzing a NP suspension in 50 103 M PBS with or without 10 103 M DTT at 37 8C. Briefly, 1 mL of NP suspension was put in a dialysis tube (MWCO 12 000) and dialyzed against both types of media, i.e., PBS and PBS þ DTT media. After certain time intervals, 6 mL of release media were taken out and refilled with an equal volume of fresh medium. 1 mL of DCM was added to the release medium, shaken well, and separated the organic layer. Later, the organic layer was mixed with 5 mL of a mixture of methanol, water, and acetonitrile, and DCM was evaporated completely by purging with N2 gas. The released amount of PTX in the resulting solution was determined by HPLC. The release experiments were conducted in triplicate and the results presented here are the average data.
2.7. Cell Viability Assay MCF-7 breast cancer cells were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin at 37 8C in a humidified environment of 5% CO2. Before performing the experiments, the cells were pre-cultured until confluence was reached. The 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to study the cytotoxicity of PCL-PEI-FA/PCL-S-S-PEG NPs against MCF-7 breast cancer cells. The cells were seeded in 96-well plates at 4 104 cells ml1. After 24 h incubation, the medium was replenished with PCLPEI-FA/PCL-S-S-PEG NP suspensions of different concentrations. The cells were incubated for 24, 48, and 72 h, respectively. After this, wells were washed thrice with PBS. 10 mL of freshly prepared MTT solution and 90 mL of medium were added to each well. After incubating for approximately another 4 h, MTT medium solution was removed leaving a precipitate. 100 mL of DMSO were added to each well and the plate was shaken gently in order to dissolve the precipitate. The absorbance of MTT at 570 nm was observed using a microplate reader after subtracting the absorbance of the corresponding control wells. Cell viability was represented as the ratio of the absorbance of the cells incubated with NP suspension to that of the cells incubated with culture medium only (Equation (2)). Cell Viability ¼
Absorbance of cells treated with drug loaded NPs Absorbance of untreated cells 100
ð2Þ
2.8. Cellular Uptake MCF-7 breast cancer cells were cultured in the confocal imaging chambers (LAB-TEK, Chambered Coverglass System) at 37 8C. The
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of the drug loaded in the final product (Equation (1)).
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medium was changed after an appropriate interval until 80% confluence was reached. The medium was removed and cells were washed thrice with PBS. Coumarin-6 loaded NPs were then placed into the confocal imaging chamber, and cells were incubated for 8 h. Later, cells were washed with PBS thrice, fixed with 75% ethanol for 20 min and further washed with PBS twice. Finally, the cells were imaged using a Confocal Laser Scanning Microscope (CLSM, Zeiss LSM 410, Jena, Germany) with imaging software (Fluoview FV1000).
2.9. Characterization FTIR was carried out using a Bruker IFS 66 V vacuum-type spectrometer in a range between 4000 and 500 cm1 by mixing freeze-dried copolymer powder with KBr. 1H NMR was carried out using a Bruker MSL-300 spectrometer with tetramethylsilane as an internal standard. GPC was conducted utilizing a Waters 244 gel permeation chromatograph instrument with tetrahydrofuran as the eluent at a flow rate of 1 mL min1. UV-vis spectroscopy was used to obtain the folic acid content of PCL-PEI-FA on the basis of a standard curve. PCL-PEI-FA polymer solution and FA solutions of different concentrations were prepared. The absorbance of these solutions was measured from 200 to 500 nm with a Shimadzu UV-3600 spectrophotometer and the FA content was determined at 280 nm. The particle sizes of PCL-PEI-FA/PCL-S-S-PEG NPs were determined by the dynamic laser scattering (DLS) technique (Brookhaven Instruments Corporation 90 plus Particle Sizer, USA) at 25 8C with at a scattering angle of 908. The zeta potential of the NPs was measured using laser Doppler anemometry (Zeta Plus, Zeta Potential Analyzer, Brookhaven Instruments Corporation, USA). Prior to zeta potential calculations, the NP suspension was diluted in de-ionized water. The value was recorded as the average of three measurements. The morphology of the NPs was observed by scanning electron microscopy (FESEM, JEOL JSM- 6700F, Japan) and transmission electron microscopy (TEM, JEOL TEM-100, Japan). The size change in the micelles, due to destabilization and agglomeration, in response to DTT in PBS was monitored using DLS measurements. Briefly, 10 103 M DTT solution was obtained by mixing 1.5 mL of PCL-PEI-FA/PCL-S-S-PEG NP suspension and DTT in 50 103 M PBS. It was then placed in a shaking bath with a speed of 200 rpm at 37 8C. The micelle size was measured at appropriate intervals using the DLS technique.
3. Results and Discussion 3.1. Synthesis and Analysis of Copolymers A conjugation technique was utilized to synthesize PCL-PEIFA and PCL-S-S-PEG block copolymers. The carboxylic acid group of PCL can be coupled to the primary amine of PEI using DCC and NHS conjugation agents.[20] In brief, the reaction of the carboxylic acid of PCL with NHS and DCC produces DCC activated ester. These activated esters can be coupled with PEI to form stable conjugated PCL-PEI copolymer.[21] Since PEI has amine groups attached at both
ends, an excess amount of PEI was used in order to prohibit the synthesis of PCL-PEI-PCL.[22] Similarly, FA was activated via DCC and NHS and thereafter conjugated with PCL-PEI to produce PCL-PEI-FA copolymer, as shown in Scheme 2. Disulfide containing copolymer was synthesized by the oxidation reaction of thiols to yield disulfide containing PEG, and it was then conjugated with PCL. PTAD is considered to be an efficient reagent for the oxidation of thiols to their respective disulfide compound.[23] Herein PEG-SH was reacted with cysteamine using PTAD to obtain PCL-cysteamine, and later PCL-cysteamine was coupled with activated PCL to produce PCL-S-S-PEG (Scheme 2b). Figure 1 shows the FTIR spectra of PCL, PCL-PEI-FA, PEGthiol, and PCL-S-S-PEG. PCL shows the representative peaks of carbonyl groups at 1720 cm1, C—H peaks of aliphatic groups at 2947 cm1 and 2865 cm1, and carbon-oxygen stretching at 1170 cm1.[24] In the spectrum of PCL-PEI-FA, the characteristic peaks of amine at 3321 cm1 and 1550 cm1, and the carbonyl group peak of amide at 1650 cm1 describe the successful coupling of PCL with PEI.[25] The representative peaks of PEG at 2850–3000 cm1 for CH2 groups, 1380 cm1 for C—H bending, and 1100 cm1 for C—O—C stretching, along with the peaks of the SH group at 2505 cm1 were obtained in the PEG-thiol spectrum.[26] However, the SH group peak vanishes and a new peak for CH2—S originates at 1270 cm1 in PCL-S-S-PEG copolymer.[27] The representative peaks of PCL and the carbonyl peak of amide at 1650 cm1 were also observed in the PCL-S-S-PEG spectrum, which clearly indicates the formation of PCL-S-S-PEG. The chemical structures of these synthesized copolymers were also verified by 1H NMR, as shown in Figure 2. Figure 2a shows the 1H NMR spectrum of PCL-PEI-FA, which demonstrates the four major characteristic peaks of PCL at 1.4 (c), 1.6 (b), 2.3 (a), and 4.1 (d) ppm, attributed to the repeating units of PCL.[28] The peak at about 2.7 (e) ppm is related to PEI,[29] and the characteristic peaks of FA at 6.5– 7.0 (f) ppm can be clearly observed. PCL-S-S-PEG also showed characteristic peaks of PEG at about 3.7 and 3.4 ppm, and the peaks of disulfide linkages at about 2.85 and 2.92 ppm (Figure 2b).[30] Based on this data from 1H NMR, together with the results from FT-IR, it seems safe to claim that PCL-PEI-FA and PCL-S-S-PEG were successfully obtained using the conjugation technology. The content of FA in the PCL-FEI-FA has a great effect on its targeting ability to the tumor. Thus, the crosslinking of FA with PCL-PEI and its content were investigated, with the UV-vis spectrum of PCL-PEI-FA shown in Figure 3. The characteristic absorbance peak of FA at 360 nm was observed both in PCL-PEI-FA and the standards, which confirmed the successful conjugation of FA onto PCL-PEI copolymer.[31] Later, the FA content in the PCL-PEI-FA was determined by the standard curve method and was about 0.019 mg mL1. This value indicated that 96% (mol) of FA
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Scheme 2. Chemical pathways for copolymer synthesis.
was conjugated onto PCL-PEI, calculated from the feeding amount. The content of FA in the polymer could also be controlled through changing the feeding amount. The molecular weight of these obtained copolymers was measured with GPC, which is shown in Figure 4. After
the conjugation, narrow molecular weight distributions of copolymers with M n of 14 100 Da and 12 200 Da for PCL-S-SPEG and PCL-PEI-FA were observed, respectively. Considering the molecular weight of the original polymer (PCL, 10 000 Da, PEG-SH, 2000 Da), we thought that PCL-S-S-PEG
Figure 1. FTIR spectra of (a) PCL, (b) PCL-PEI-FA, (c) PEG-thiol and (d) PCL-S-S-PEG.
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Figure 2. 1H NMR spectra of (a) PCL-PEI-FA and (b) PCL-S-S-PEG.
Figure 3. UV-vis spectra of (a) PCL-PEI-FA with standard concentrations of FA and (b) their standard curve.
was successfully obtained. The polydispersity index (PDI) was 1.57 for PCL-S-S-PEG and 1.32 for PCL-PEI-FA. The single peaks of copolymers, as shown in Figure 4, show the monodispersity of the molecular weights of the copolymers and the absence of PCL-PEI-PCL formation during the conjugation reaction. 3.2. The Size and Morphology of PCL-PEI-FA/PCL-S-SPEG NPs Copolymers with both hydrophobic and hydrophilic parts normally assemble into core-shell type NPs in aqueous solution due to their amphiphilic nature.[32] Similarly, in
this work, core-shell nanoparticles were obtained by mixing two kinds of amphiphilic copolymers, i.e., PCLPEI-FA and PCL-S-S-PEG. The surface morphology of PCLPEI-FA/PCL-S-S-PEG NPs was characterized with FESEM, as shown in Figure 5a. The shape and size of these NPs was investigated by TEM (Figure 5b). PCL-PEI-FA/PCL-S-S-PEG NPs showed a spherical shape with a uniform size of approximately 230 nm. Their size and size distribution were further investigated using DLS. Figure 5b shows DLS data for PCL-PEI-FA/PCL-S-S-PEG NPs in aqueous medium. It was observed that the NPs exhibited a unimodal size distribution and the average NP size obtained from DLS data was about 270 nm. This size is a little bigger than
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Figure 4. GPC chromatograms of copolymers.
that of FESEM, because DLS gives the hydrodynamic mean diameter while FESEM provides the mean diameter in the dry state. Figure 6a describes the zeta potential trend of PCL-PEI-FA/ PCL-S-S-PEG NPs with an increasing proportion of PCL-PEIFA copolymer. When the mole ratio of PCL-PEI-FA in the NPs increased from 5% to 20%, the zeta potential of PCL-PEI-FA/ PCL-S-S-PEG NPs increased from 2.2 mV to 7.1 mV. This change in zeta potential is reasonable because PCL-PEI-FA is a positively charged polymer due to the PEI segment. Thus, increasing the ratio of PCL-PEI-FA copolymer causes an increment in the zeta potential of these NPs. Positive zeta potential is an essential requirement for the endocytosis of NPs by tumor cells due to the electrostatic attraction between positively charged NPs and negatively charged cell membranes.[33] Based on the above fact, the ratio of PCL-PEI-FA could be adjusted to be as high as possible in order to get effective binding ability.
Figure 5. SEM image of PCL-S-S-PEG/PCL-PEI-FA NPs with their surface morphology (inset) (a); TEM image of NPs (b); DLS result for NPs in aqueous solution (c).
Figure 6. Zeta potential of NPs (a) and size of NPs (b) with different proportions of PCL-PEI-FA to PCL-S-S-PEG.
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The size of PCL-PEI-FA/PCL-S-S-PEG NPs with different ratios of PCL-PEI-FA to PCL-S-S-PEG was measured by DLS and is shown in Figure 6b. It can be clearly observed that PCL-PEI-FA/PCL-S-S-PEG NPs enlarged when the amount of PCL-S-S-PEG copolymer was increased. Since PCL-PEI-FA/ PCL-S-S-PEG NPs are composed of a core-shell structure, where PCL is the core and PEG is the shell, increments in the proportion of PCL-S-S-PEG copolymers in the NPs result in thickening of the outermost PEG layer. As a result, the size of NPs increases. 3.3. Reduction Triggered Destabilization Cancer cells possess an internal reductive environment due to a high level of presence of antioxidant-Glutathione.[34] In order to obtain an in vitro reductive environment similar to cancer cells, DTT is commonly used to obtain reductioninduced degradation. The disulfide bond is cleavable in the presence of a reducing agent such as DTT. Herein, DTT reduces the disulfide linkages of PCL-S-S-PEG to thiols via two sequential thiol-disulfide exchange reactions.[35] After the cleavage of the disulfide bond of PCL-S-S-PEG, PCL-SH and PEG-SH are formed. The PEG-SH would detach from the PCL-PEI-FA/PCL-S-S-PEG NPs, exposing targeting ligands. This overall phenomenon will cause destabilization and the aggregation of NPs, which will improve the NPs’ targeting ability to tumor cells and stimulate the uptake of the NPs by tumor cells. To test this point, 10 103 M DTT in 50 103 M PBS at pH 7.4 was used to mimic the reductive physiological environment and facilitate the reduction-induced destabilization of the disulfide bonds of PCL-S-S-PEG. The size change of PCLPEI-FA/PCL-S-S-PEG NPs in response to DTT was investigated by DLS, as shown in Figure 7. The average particle size of PCL-PEI-FA/PCL-S-S-PEG NPs remained constant in the absence of DTT during the testing period. However, after the addition of DTT, the size of PCL-PEI-FA/PCL-S-SPEG NPs increased from 270 nm to about 1000 nm within 60 min, which clearly confirmed that the PEG segments were detached from the nanoparticles, resulting in particle aggregation and break up of the NPs. Moreover, this destabilization of the NPs was enhanced with an increase in the proportion of PCL-S-S-PEG copolymer in the NPs, since a higher amount of PCL-S-SPEG was conducive to a higher detachment rate of PEG and more serious particle aggregation. 3.4. Drug Encapsulation and Drug Release Behavior of NPs Core-shell NPs with a hydrophobic interior and a hydrophilic exterior environment are promising candidates for encapsulating hydrophobic drug in their cores, while the hydrophilic shell stabilizes NPs in aqueous media.[36]
Figure 7. Size change of PCL-PEI-FA/PCL-S-S-PEG micelles in response to DTT measured by DLS.
Herein, PTX was selected as a model drug to encapsulate inside these PCL-PEI-FA/PCL-S-S-PEG NPs. The drug loading ability and in vitro release profiles are depicted in Figure 8. Figure 8a describes the PTX encapsulation efficiency of NPs against the PCL-PET-FA mole ratio inside the NPs. It was found that as the ratio of PCL-PEI-FA increased, the PTX encapsulation efficiency decreased. We thought PEG, being a hydrophilic shell, played a vital role in the stabilization of the NPs. A high proportion of PCL-S-S-PEG copolymer in the NPs enhanced the stability of PCL-PEI-FA/PCL-S-S-PEG NPs, and gave high yields of NPs. After increasing the ratio of PCL-PEI-FA, the stability of PCL-PEI-FA/PCL-S-S-PEG NPs decreased; more PTX loaded NPs would congregate and form large aggregates, which would be filtered out during the preparation procedure. Thus lower drug loading efficiency was obtained. The drug release profile of PTX loaded NPs with and without DTT is shown in Figure 8b. For all samples with different polymer ratios, less than 8% of encapsulated PTX was released from the PCL-PEI-FA/PCL-S-S-PEG NPs within 20 h, which indicated that PCL-PEI-FA/PCL-S-S-PEG NPs encapsulated PTX very well and provided a sustained drug release profile. However, in the presence of DTT, an abrupt release of PTX was found and up to 82% of PTX was released out from the NPs in 20 h. Moreover, the PTX released from NPs with a high proportion of PCL-S-S-PEG was greater when compared to those with a low proportion. The split of the disulfide bonds causes the detachment of PEG and the structural breakdown of the NPs, which consequently enables a quick release of PTX. As the proportion of PCL-S-S-PEG increased, more PCL-S-S-PEG involved inside the NPs would break up due to DTT.
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Figure 8. PTX encapsulation efficiency of NPs with different ratios of copolymers (a) and PTX release behavior of NPs with and without DTT (b).
Therefore, the break up rate of the NPs is accelerated, which enables a faster drug release. 3.5. Cell Viability and Cellular Uptake of NPs The cytotoxicity of drug loaded NPs is greatly related to the binding and cellular uptake capabilities of the NPs to the targeting cells. In this work, the PEG chain of PCL-PEI-FA/ PCL-S-S-PEG NPs shields the positively charged PEI segment, and also protects the FA ligand from contact with the biomacromolecules before the NPs reach the targeting site. At the tumor site, the PEG layer detaches from the NPs, and PEI segments and FA ligands are exposed to the tumor cells directly. The positively charged PEI segment will enhance the binding ability of NPs with tumor cells due to the static electronic interaction.[37] The presence of FA will boost the tumor cells’ selective uptake of these NPs because there are many FA receptors on the surface of tumor cells. In order to evaluate the cytotoxicity of PCL-PEI-FA/PCL-S-S-PEG NPs, the viability of the MCF-7 cell line was tested in the presence of empty and drug loaded NPs. Figure 9 describes the cytotoxicity of empty and PTX loaded NPs incubated with MCF-7 cells for 24, 48, and 72 h using the MTT assay. It apparently showed that empty NPs had very little toxicity against MCF-7 cells, whereas PTX loaded NPs exhibited noticeable cytotoxicity with increasing PTX concentration. Moreover, the toxicity of drug loaded NPs increased with the passage of time. This could be attributed to the sustained release of PTX from NPs. After incubation for 72 h, the highest cytotoxicity of PTX loaded NPs was observed. This implies that the amount of the PTX released
Figure 9. Cell viability of MCF-7 cells incubated with blank and PTX loaded NPs.
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from the NPs is high enough to suppress the proliferation of MCF-7 cells. It has been reported that stealthy NPs with specific targeting characteristics can escape from endosomes and be delivered to cytoplasmic organelles.[38] The fate and the cellular uptake of Coumarin-6 loaded PCL-PEI/PCL-S-S-PEG NPs and Coumarin-6 loaded PCL-PEI-FA/PCL-S-S-PEG NPs was studied by CLSM incubated with the MCF-7 cell line for 6 h. Coumarin-6 showed green fluorescence in intracellular compartments such as cytosol and cell nucleus after 6 h
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Figure 10. CLSM of MCF-7 cells incubated for 6 h with coumarin-6 loaded PCL-PEI/ PCL-S-S-PEG NPs (a) and PCL-PEI-FA/PCL-S-S-PEG NPs (b).
incubation with both types of NPs, which indicated the rapid engulfment of NPs and the release of Coumarin-6 inside cells (Figure 10). The disulfide bond is broken in the intracellular compartments because of the relatively high concentration of reducing glutathione tripeptide,[39] which could accelerate intracellular distribution of coumarin-6.
4. Conclusion In order to fabricate multifunctional NPs for cancer therapy, two types of copolymer were synthesized via conjugation techniques; PCL-PEI-FA contains specific targeting characteristics while PCL-S-S-PEG possesses a long circulation property. Furthermore, the disulfide linkage of PCL-S-S-PEG was utilized to generate reduction-induced destabilization capabilities in these NPs. By adjusting the ratio of these copolymers, core-shell structured NPs of approximately 230 nm were obtained. These core-shell structured NPs showed enhanced stability and PTX encapsulation efficiency. However, these NPs exhibited quick PTX release in a reductive environment, which improved their effectiveness against tumors. Moreover, the long circulation and
specific targeting qualities of PEG and FA embedded in the outer layer could make this system an efficient delivery platform for anticancer drugs.
Acknowledgements: This work was supported by the National Natural Science Foundation of China (No. 21105047 and 51173077), and Fundamental Research Funds for the Central Universities (1085021309).
Received: May 8, 2014; Revised: July 12, 2014; Published online: DOI: 10.1002/mabi.201400228 Keywords: core-shell structures; long circulation; nanoparticles, polycaprolactone; reduction induced degradation
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