www.advmat.de www.MaterialsViews.com
COMMUNICATION
Fabrication of a Multifunctional Nano-in-micro Drug Delivery Platform by Microfluidic Templated Encapsulation of Porous Silicon in Polymer Matrix Hongbo Zhang,* Dongfei Liu, Mohammad-Ali Shahbazi, Ermei Mäkilä, Bárbara Herranz-Blanco, Jarno Salonen, Jouni Hirvonen, and Hélder A. Santos* Orally administrated targeted drug delivery systems (TDDS) to the desired location in the gastrointestinal (GI) tract are highly demanded for the treatment of local diseases, such as colorectal cancer.[1] However, due to the harsh conditions and manifold barriers of the GI tract, many drugs are hindered from reaching the targeted site.[2] Uniform particle size is critical for these drug delivery systems, since it might significantly affect the biological interactions of the payloads in vivo.[3] An ideal advanced TDDS should efficiently protect the drugs in the varying GI conditions and precisely release the drugs at the targeted site. TDDS should have preferably high drug loading capacity, with the possibility to load multiple drugs with different physicochemical properties, especially for the treatment of complex diseases like cancers. Moreover, mucoadhesive properties of the TDDS are highly preferable to prolong the retention time of the drugs at the site of absorption, thus improving the therapeutic efficacy and bioavailability. Droplet-based microfluidics is a promising technique that can be applied to manipulate nanoliter volumes of immiscible carrier phases in the networks of micron scale channels and precisely dispersing these solutions into equally sized microdroplets; subsequently, the droplets can be solidified to form monodisperse microparticles.[3,4] The advantages of the microfludic technique are the possibility of fabricating monodisperse particulate structures, as well as the flexibility in material selection with an encapsulation efficiency of almost 100%.[3,4] Porous silicon (PSi) has been intensively investigated in drug delivery applications.[5] This material has unique properties such as high surface-to-volume ratio, high drug-loading capacity and the potential to deliver chemical drugs with multiple structures, proteins, siRNA, and viruses.[5d,6] Moreover, Dr. H. B. Zhang, D. F. Liu,[+] M. A. Shahbazi,[+] E. Mäkilä, B. Herranz-Blanco, Prof. J. Hirvonen, Prof. H. A. Santos Division of Pharmaceutical Chemistry and Technology Faculty of Pharmacy University of Helsinki Helsinki, FI-00014, Finland E-mail: [email protected]; [email protected] E. Mäkilä, Prof. J. Salonen Laboratory of Industrial Physics Department of Physics University of Turku Turku, FI-20014, Finland [+]These authors contributed equally to this work
DOI: 10.1002/adma.201400953
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
PSi has not only a good biocompatibility and biodegradability, but also the PSi surface can be chemically modified to present reactive groups for subsequent conjugation with different polymers, fluorescent probes, contrast agents, and biologically active targeting moieties.[5d,7] All these advantages make the PSi particles an ideal choice for drug delivery and biomedical applications. Typical mucoadhesive polymers have active groups capable to form strong hydrogen bonds, together with appropriate high molecular weight and density for cross-linking.[2] Among the mucoadhesive polymers, poly(methyl vinyl ether-co-maleic acid) (PMVEMA) polymer was selected for this study due to its overall favorable properties.[8] Herein, we conjugated the PMVEMA with carboxylic acid modified thermally hydrocarbonized porous silicon nanoparticles (PSi NPs) using polyethyleneimine (PEI) as a linker polymer. PEI was introduced due to its high density of amine groups to facilitate the conjugation of PMVEMA. In addition, the positively charged amine groups of PEI can also aid in enhancing the cellular interactions of the produced particles.[9] Subsequently, the microfluidic technique was applied to fabricate multistage TDDSs by encapsulating the PSi-PEI-PMVEMA inside a pH-responsive hydroxypropylmethylcellulose acetate succinate based polymer (ASHF).[10] The PSi NPs (average particle size of 151.1 ± 4.0 nm and zeta-potential of −36.0 ± 0.6 mV) were fabricated using electrochemical anodization, followed by thermal hydrocarbonization, surface modification with undecylenic acid and milling as described elsewhere.[11] PEI and PMVEMA were conjugated to the PSi NPs via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemical reaction to form PSi-PEI-PMVEMA (average particle size of 366.7 ± 3.0 nm and zeta-potential of −34.3 ± 2.0 mV) (Figure 1a).[8a] The success of the polymer conjugation was evaluated by Fourier transform infrared spectroscopy (FTIR), where the characteristic peaks from the PSi, PEI, and PMVEMA were also detected from the PSi-PEI-PMVEMA spectrum (Figure S1a). The PSi-PEI-PMVEMA NPs were subsequently encapsulated in ASHF to form the nano-in-micro composites, PSiPEI-PMVEMA@ASHF (average particle size of ca. 30 µm), using a flow focusing microfluidic chip for producing an oilin-water (O/W) emulsion (Figure 1a). Ethyl acetate (EA) was used as a non-toxic organic solvent, which has fast diffusion rate to water due to its high solubility.[12] These properties are beneficial for producing particles for drug delivery applications with good stability and high encapsulation efficiency.[12] Thus,
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
1
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
Figure 1. (a) Schematic illustration of the fabrication process of the PSi-PEI-PMVEMA@ASHF composites. The PSi-PEI-PMVEMA NPs were prepared by conjugation with the PEI and PMVEMA polymers. Then, the PSi-PEI-PMVEMA NPs were encapsulated in the pH-responsive ASHF polymer with microfluidics. The 5-FU@PSi-PEI-PMVEMA NPs were dispersed in the inner fluid together with ASHF and CEL in EA. The outer continuous fluid contained 2% of P-407. (b) The monodispersity of the PSi-PEI-PMVEMA NPs of the fabricated PSi-PEI-PMVEMA@ASHF composite were studied by confocal microscope and SEM. The PSi-PEI-PMVEMA NPs were conjugated with FTIC and the ASHF layer was labeled with TRITC. The confocal images showed (from left to right) the FITC, TRITC and the overlay channels. In addition, the surface of the particles was monitored by SEM. (c) Dissolution behavior of the PSi-PEI-PMVEMA@ASHF composite at pH 7.4 monitored from 2 to 30 min by confocal microscopy; all images presented are the overlay channels.
PSi-PEI-PMVEMA along with the ASHF in EA solution was used as the inner fluid. A non-toxic amphiphilic Poloxamer 407 (P-407) was dissolved at pH 5.5 and was used as the outer fluid due to its ability to stabilize the formed droplets. In addition, the hydrophilic fluorouracil (5-FU) and the hydrophobic drug celecoxib (CEL), which act synergistically for colon cancer,[13] were loaded inside the composite. Due to the distinct physicochemical properties of both drugs, it is very challenging to first encapsulate and subsequently release the drugs from one single formulation. CEL has good solubility in EA and it was directly dissolved in this solvent. PSi-PEI-PMVEMA NPs were introduced within the ASHF polymer matrix to improve the drug loading capacity of the produced composite. By loading the 5-FU inside the PSi-PEI-PMVEMA NPs and then encapsulating the drug-loaded NPs inside the ASHF polymer, we 2
wileyonlinelibrary.com
were able to increase the loading degree of 5-FU from 0.5% to 7.1% (w/w). Moreover, the loading ratios of CEL to 5-FU could be freely adjusted. Herein, we tailored the drug loading ratio to approximately 1:1 by adjusting the loading degree of CEL to 6.9% (w/w). The success of the drugs encapsulation was confirmed by FTIR (Figure S1b). The flow rates in the two channels of the microfluidic chip were optimized in order to obtain particles with the desired size and size distribution. The inner fluid was fixed at 1500 µL/h and the outer fluid was adjusted to the flow at a rate of 15000 µL/h. Thus, we were able to obtain a particle size of ca. 50 µm by jetting regime (Figure S2). By decreasing the flow rate of the outer fluid from 15000 µL/h to 6000 µL/h, the flow changed from jetting to dripping regime (Figure S2), and the particle size decreased to ca. 30 µm (Figure 1b) with improved
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
www.advmat.de www.MaterialsViews.com
COMMUNICATION Figure 2. (A) Dissolution behavior of the PSi-PEI-PMVEMA@ASHF composite at different pH-values. The SEM images show the apparent morphology and surface properties of the particles after 2 h at different pH-conditions. By incubating at pH 7.4 for 2 h, the spherical composite totally dissolved and in the image we can only observe a polymer matrix. (B) Drug release profiles of 5-FU and CEL from the PSi-PEI-PMVEMA@ASHF composites in a series of pH buffers at 37 °C. The mixture of free 5-FU and CEL (ratio 1:1) was used as control. Data represent mean ± s.d. (n = 3).
(narrow) size distribution (Figure S2). These results are in good agreement with the previous findings that a dripping regime is more favorable in producing monodisperse emulsions.[14] The drops were collected in 2% P-407 at pH 5.5 (same as the outer fluid) and the droplets were solidified by diffusion of the EA to the outer aqueous phase. Due to the high water solubility of EA, fast solidification process could be achieved and can be further accelerated by frequently changing the outer aqueous solution, which will significantly improve also the monodispersity of the fabricated particles.[12] After drying in vacuum oven, the particles were collected as powders, and remained stable during and after long-term storage (results not shown). Since no toxic solvents were used during the production, the obtained particles can be administered orally. The monodispersity of the produced PSi-PEI-PMVEMA@ ASHF composites was evaluated by confocal microscopy, particularly the size distribution of the composites and the PSi-PEIPMVEMA NPs distribution within the composites. To enable the visualization, the PSi-PEI-PMVEMA NPs were conjugated with fluorescein isothiocyanate (FITC, green) and the outer ASHF polymer layer was labeled with tetramethylrhodamine (TRITC, red). The confocal images indicated that the PSi-PEIPMVEMA NPs were very well dispersed within the PSi-PEIPMVEMA@ASHF composites (Figure 1b). In addition to the confocal imaging, scanning eletron microscopy (SEM) images showed that the composites had smooth surfaces and spherical structures (Figure 1b). Furthermore, the efficient encapsulation of the PSi-PEI-PMVEMA NPs within the ASHF polymer droplets can be observed in Figure S2 and in Video S1. The dissolution behavior of the PSi-PEI-PMVEMA@ASHF composites at pH 7.4 was observed by confocal microscopy (Figure 1c). The composite’s outer layer, ASHF, started to dissolve immediately and the PSi-PEI-PMVEMA NPs (green) were released from the composites after 2 min of incubation at pH 7.4 (Figure 1c). The composites were fully collapsed after
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
30 min (Figure 1c). In addition, the pH-responsive properties of the composites were monitored with SEM by incubating them at pH 1.2, 5.5 and 6.5 for 2 h, and at pH 7.4 for 5 min and 2 h (Figure 2a). The composites showed clear pH-responsive properties, presenting a stable structure at pH ≤ 6.5 and dissolving fast at pH 7.4 (Figure 2a). The surface topology of the composites before and after dissolution is shown in Figure S3. To further evaluate the pH-responsive properties of the PSiPEI-PMVEMA@ASHF composites, the drug release kinetics of 5-FU and CEL from the composites were studied by incubation in a series of pH buffers, starting from pH 1.2 to pH 5.5 and 6.5 for a time period of 2 h for each pH-value, and then at pH 7.4 for 8 h in order to mimic the intestinal and colonic pH and transit time of the GI tract. Since CEL is insoluble in water, 5% of P-407 was added into the release buffers. The results showed that 5-FU and CEL were almost completely dissolved after 1 and 2 h at pH 1.2. The composites successfully encapsulated 5-FU and CEL within the ASHF polymer layer, which showed no drug release at pH ≤ 6.5 (Figure 2b). By contrast, at pH 7.4, after the ASHF polymer layer was dissolved, both 5-FU and CEL were completely released within 2 h (Figure 2b). The results indicated that the drug release from the composite was clearly controlled by the outer ASHF polymeric layer. Owing to the advantages of the microfluidic technique, by changing the ASHF polymer with different pH/temperature/enzyme responsive polymers, our platform has the potential to be used in different kinds of therapeutic applications. In addition to the efficient and controlled drug delivery properties of the aforementioned composites, the TDDS should also overcome the mucus layer of the intestine, which plays an important role in protecting the epithelial cells in the GI tract, and thus, creating a barrier for drug absorption.[2] In order to enhance the therapeutical efficiency of the drugs, mucoadhesive drug formulations are often needed.[2] To achieve this, herein the PSi NPs were conjugated with PEI and PMVEMA,
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
3
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
and the effect of the mucoadhesivity was tested in Caco-2/HT-29 co-culture monolayers. Different particles were incubated with Caco-2/HT-29 monolayers for 6 h at 37°C, and at pH 7.4. In all samples, the morphology of the Caco-2/HT-29 monolayers was not affected during incubation (Figure 3a), which indicates that these particles are not toxic to Caco-2/HT-29 monolayers under the experimental conditions tested. Clear differences on the mucoadhesion between PSi and PSi-PEI-PMVEMA NPs were observed; PSi-PEI-PMVEMA NPs were observed on top of the microvilli and almost no PSi NPs were detected (Figure 3a). This proved the fact that the mucoadhesion properties of PSiPEI-PMVEMA NPs were due to the surface modification with PEI and PMVEMA. In addition, the ASHF encapsulation did not affect the mucoadhesion of PSi-PEI-PMVEMA NPs since the PSi-PEI-PMVEMA NPs released from the composites at pH 7.4 also adhered to the surface of the Caco-2/HT-29 monolayers (Figure 3a).
The mucoadhesion and cellular interactions of the PSi-PEIPMVEMA@ASHF composites were further investigated in the presence of drugs. The 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composite was incubated with Caco-2/HT-29 monolayers at the pH-values of 6.5 and 7.4. At pH 6.5, no PSi-PEI-PMVEMA NPs were observed in the TEM images. In addition, the morphology of the Caco-2/HT-29 monolayers was not affected (no release of drugs, hence no toxicity to the cells) (Figure 3b), which further proved the successful encapsulation of the payloads (PSiPEI-PMVEMA NPs and the drugs) within the composite. At pH 7.4, the ASHF polymer layer was dissolved and the drugs and PSi-PEI-PMVEMA NPs were released (Figure 3b). The PSiPEI-PMVEMA NPs adhered to the Caco-2/HT-29 monolayers, and few PSi-PEI-PMVEMA NPs were taken up and transported to the basolateral side of the Caco-2/HT-29 monolayers (Figure 3b). Since PEI has the ability to improve the cellular internalization and endosomal escape,[9] the cellular uptake and
Figure 3. (a) Mucoadhesion properties of the PSi and PSi-PEI-PMVEMA NPs, as well as PSi-PEI-PMVEMA NPs released from the PSi-PEI-PMVEMA@ ASHF composites at pH 7.4. Particles at a concentration of 100 µg/mL were incubated with Caco-2/HT-29 monolayers at pH 7.4 and 37 °C for 6 h. (b) Cellular interaction of the 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites with Caco-2/HT-29 monolayers at the pH-values of 6.5 and 7.4. All scale bars in (a) and (b) are 500 nm. (c) Drug permeation profiles across Caco-2/HT-29 monolayers. The initial drug concentration in apical side was 100 µg/mL for 5-FU and CEL. The 5-FU&CEL@PSi-PEI-PMVEMA-ASHF concentrations were calculated similarly to the pure drugs based on the drug loading degree. The cells were exposed to the bare drugs or drug-loaded PSi-PEI-PMVEMA@ASHF composites at pH 7.4 and 37 °C for 3 h. Data represent mean ± s.d. (n = 3). The level of significances between the permeability of pure drugs and 5-FU&CEL@PSi-PEI-PMVEMA-ASHF were set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001.
4
wileyonlinelibrary.com
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
www.advmat.de www.MaterialsViews.com
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
COMMUNICATION
drug transport may have been due to the presence of PEI on the surface of the PSi NPs, and disruption of the cell monolayer when in contact with the drugs (no cell morphology disruption and particle uptake was observed in the absence of drugs, as shown in Figure 3a). The distinct different behaviour of the loaded drugs in the composites at the pH-values of 6.5 and 7.4 showed that the composites had the ability to protect the payloads from premature release and ensured successful control of drug release. These properties will significantly decrease the adverse drug’s effects and enhance the therapeutic efficiency of the drug(s). The effect of the PSi-PEI-PMVEMA@ASHF composite on the drug permeation across Caco-2/HT-29 cell monolayers was another important parameter to be evaluated for the oral drug delivery systems.[15] Herein, we also investigated the permeation of 5-FU and CEL across Caco-2/HT-29 cell monolayers before and after loading them into the composites. The results indicated that the permeability of both 5-FU and CEL were enhanced by loading them into the composites (Figure 3c). The apparent permeability coefficients (Papp) of 5-FU and CEL were increased from 1.30 to 1.62 (10−5 cm/s) and 0.67 to 0.91 (10−5 cm/s), respectively. In addition, the change of the transepithelial electrical resistance (TEER) of Caco-2/HT-29 monolayers during the drug permeation experiments was also monitored, which may help in predicting the paracellular permeability of the drugs.[15a] The results showed that the mixture of the pure drugs had a clear effect on reducing the TEER-values of the Caco-2/HT-29 monolayers (Figure S4), probably induced by the cytotoxicity of the drugs. This is in good agreement with the TEM images, which showed that in the presence of drugs the PSi-PEI-PMVEMA NPs were taken up and transported to the basoleteral sites of the Caco-2/HT-29 monolayers (Figure 3b). Interestingly, we observed more extensive TEER reduction of the 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites. This is because after the dissolution of the ASHF polymer layer, the PSi-PEI-PMVEMA NPs increased the mucoadhesivity and enhanced the release of the drugs in the vicinity of the monolayers, thus enhancing also the drug permeability. In order to assess the safety of the PSi-PEI-PMVEMA@ ASHF composites, we also tested the cell viabilities of Caco-2 and HT-29 cells at the pH-values 6.5 and 7.4 (Figure S5) after incubation with the different particles. At pH 6.5, the composites showed no significant toxicity at all the concentrations tested (25 to 250 µg/mL), whereas both PSi and PSi-PEIPMVEMA NPs showed some toxicity to the cells, especially at higher concentrations (Figures S5a and S5c). At pH 7.4, the ASHF polymer layer of the composites was dissolved and the PSi-PEI-PMVEMA NPs were exposed to the cells. As a result, only a slight difference on the cell viability was observed between the PSi-PEI-PMVEMA NPs and the composites (Figure S5b and 5d). Moreover, both Caco-2 and HT-29 cells behaved similarly, which showed that the toxicity was not cell type dependent. The different behaviour of the composites on the cell viability at different pH-values can be attributed to the successful encapsulation of the PSi-PEI-PMVEMA NPs and the pH-responsive of the composites. The impact of the 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites on the cell proliferation of Caco-2 and HT-29 cells at the pH-values 6.5 and pH 7.4 was also evaluated. At both
pH-values, the pure drug combinations significantly inhibited cell proliferation (Figure 4). The composites had no significant effect on the cell proliferation at pH 6.5, which was due to the absence of drug release (Figure 4). However, the composites remarkably inhibited the cell proliferations of both cell types at pH 7.4, and the inhibition effect was even stronger than with the mixture of both pure drugs. This phenomenon could be ascribed to the high mucoadhesive property of the PSi-PEIPMVEMA NPs to the cells’ surface and to the local accumulation of the drugs in the vicinity of the cells. The proliferation results are also in good agreement with both the drug release experiments (Figure 2b) and the TEM images of the composite’s mucoadhesion properties (Figure 3b). In conclusion, we have developed here a novel PSi-PEIPMVEMA@ASHF carrier system for TDDS by microfluidics. The composite showed well-defined spherical structure, narrow size distribution, and smooth surface. The particles can be dried as free-blowing powders and re-dispersed without damaging their structure. The fabricated composites showed promising properties for targeted drug delivery, including the capacity of loading multidrugs with different physicochemical properties (with precisely adjusting the drug loading ratios
Figure 4. Cell proliferation profiles of (a) Caco-2 and (b) HT-29 cells treated with series concentrations of mixtures of 5-FU and CEL, and the corresponding concentrations of 5-FU&CEL@PSi-PEI-PMVEMA-ASHF (based on drug loading degree) at the pH-values 6.5 and 7.4. Lines A and C are 5-FU and CEL drug mixtures at the pH-values 6.5 and pH 7.4; lines B and D are 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites at the pH-values 6.5 and pH 7.4. The cells were incubated at 37 °C for 24 h in the presence of 5% P-407. Data represent mean ± s.d. (n = 3). The level of significances between the inhibition curve of the pure drugs and the drug-loaded composites under the same pH-value are set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
5
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
among the different drugs); successful encapsulation of the payloads within a pH-responsive matrix; simultaneous release of multidrugs; and controllable inhibition of the cancer cell proliferation in response to the pH changes. This TDDS was extremely stable at pH ≤ 6.5 and completely protected the payloads from premature release, but allowed fast release at pH 7.4. Furthermore, the released PSi-PEI-PMVEMA NPs showed strong mucoadhesion properties, therefore enabling the nanoin-micro composites to enhance the drug permeability and strengthen the inhibition effect on cell proliferation at pH 7.4. Owing to the properties of the composites and the flexibility of the microfluidic technique, the developed composites can be further extended as a platform for a variety of therapeutical applications.
Experimental Section The fabrication of the PSi NPs is described elsewhere.[11] PEI and PMVEMA were conjugated to PSi NPs via EDC/NHS chemical reaction to form PSi-PEI-PMVEMA (Figure 1). The success of the conjugation and characterization of the drug loading was studied by FTIR (Vertex 70, Bruker, USA), using a horizontal attenuated total reflectance accessory (MIRacle, PIKE Technologies, USA). PSi-PEI-PMVEMA was subsequently encapsulated with ASHF, using a flow focusing microfluidic chip to produce an oil-in-water (O/W) emulsion. 5-FU and CEL were chosen as model drugs; 5-FU was loaded into the PSi-PEI-PMVEMA NPs by using an immersing method as described elsewhere,[15a,16] and CEL was dissolved in the inner fluid in EA. The inner fluid contained 5-FU loaded PSi-PEI-PMVEMA NPs, ASHF and CEL in EA and the outer fluid was 2% P-407, pH 5.5 (Figure 1). The monodisperse distribution of PSi-PEI-PMVEMA NPs within the ASHF polymeric layer was studied by an inverted confocal fluorescence microscope (Leica SP5 II HCS A, Germany) and the surface morphology of the PSi-PEI-PMVEMA@ASHF composites was monitored by a Quanta 250 SEM (FEG, USA). The pH-responsive properties of the PSi-PEI-PMVEMA@ASHF composites were studied by SEM imaging after incubating the composites with different pH buffers (1.2, 5.5, 6.5 and 7.4). In addition, the dissolution of the composites at pH 7.4 was recorded with confocal microscope between 2 to 30 min. The drug loading degree and release kinetics were analyzed by HPLC (details can be found in the Supporting Information). The drug release was studied under a series of pH buffers (pH 1.2, 5.5, 6.5 and 7.4) with gradient. The mucoadhesion property of the PSi and PSi-PEI-PMVEMA NPs was studied by incubating different particles with Caco-2/HT-29 monolayers and then imaging them by TEM (Jeol JEM1400, Jeol Ltd., Japan). The drug permeability across the cell monolayers and TEER-values were also measured (see details in Supporting Information). Last, we studied the cell viability and the cell proliferation in Caco-2 and HT-29 cells at the pH-values 6.5 and 7.4 with an ATPbased CellTiter-Glo assay (Promega Corporation, USA). Further detailed experimental procedures are provided in the Supporting Information.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The financial support from the Academy of Finland (decision numbers 252215 and 256394), the University of Helsinki Funds, Biocentrum
6
wileyonlinelibrary.com
Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013, Grant No. 310892) are highly acknowledged. HARKE Pharma (Germany) is acknowledged for kindly providing the ASHF pH-responsive polymer (Shin-Etsu AQOAT). Dr. H. Zhang acknowledges the Finnish Cultural Foundation for a Post-doc Pooli grant. Received: February 28, 2014 Revised: March 23, 2014 Published online:
[1] Y. S. Krishnaiah, M. A. Khan, Pharm. Dev. Technol. 2012, 17, 521. [2] M. A. Shahbazi, H. A. Santos, Curr. Drug Metab. 2013, 14, 28. [3] W. J. Duncanson, T. Lin, A. R. Abate, S. Seiffert, R. K. Shah, D. A. Weitz, Lab Chip 2012, 12, 2135. [4] a) S. Yang, F. Guo, B. Kiraly, X. Mao, M. Lu, K. W. Leong, T. J. Huang, Lab Chip 2012, 12, 2097; b) D. Liu, B. Herranz-Blanco, E. Mäkilä, L. R. Arriaga, S. Mirza, D. A. Weitz, N. Sandler, J. Salonen, J. Hirvonen, H. A. Santos, ACS Appl. Mater. Inter. 2013, 5, 12127; c) B. Herranz-Blanco, L. R. Arriaga, E. Makila, A. Correia, N. Shrestha, S. Mirza, D. A. Weitz, J. Salonen, J. Hirvonen, H. A. Santos, Lab Chip 2014. [5] a) A. Parodi, N. Quattrocchi, A. L. van de Ven, C. Chiappini, M. Evangelopoulos, J. O. Martinez, B. S. Brown, S. Z. Khaled, I. K. Yazdi, M. V. Enzo, L. Isenhart, M. Ferrari, E. Tasciotti, Nat. Nanotechnol. 2013, 8, 61; b) E. Tasciotti, X. W. Liu, R. Bhavane, K. Plant, A. D. Leonard, B. K. Price, M. M. C. Cheng, P. Decuzzi, J. M. Tour, F. Robertson, M. Ferrari, Nat. Nanotechnol. 2008, 3, 151; c) J. H. Park, L. Gu, G. von Maltzahn, E. Ruoslahti, S. N. Bhatia, M. J. Sailor, Nat. Mater. 2009, 8, 331; d) H. A. Santos, L. M. Bimbo, V. P. Lehto, A. J. Airaksinen, J. Salonen, J. Hirvonen, Curr. Drug Disc. Technol. 2011, 8, 228. [6] a) L. M. Bimbo, O. V. Denisova, E. Mäkilä, M. Kaasalainen, J. K. De Brabander, J. Hirvonen, J. Salonen, L. Kakkola, D. Kainov, H. A. Santos, ACS Nano 2013, 7, 6884; b) D. Liu, L. M. Bimbo, E. Makila, F. Villanova, M. Kaasalainen, B. Herranz-Blanco, C. M. Caramella, V. P. Lehto, J. Salonen, K. H. Herzig, J. Hirvonen, H. A. Santos, J. Control. Release 2013, 170, 268. [7] a) C.-F. Wang, E. M. Mäkilä, M. H. Kaasalainen, D. Liu, M. P. Sarparanta, A. J. Airaksinen, J. J. Salonen, J. T. Hirvonen, H. A. Santos, Biomaterials 2014, 35, 1257; b) L. M. Bimbo, M. Sarparanta, E. Makila, T. Laaksonen, P. Laaksonen, J. Salonen, M. B. Linder, J. Hirvonen, A. J. Airaksinen, H. A. Santos, Nanoscale 2012, 4, 3184. [8] a) M.-A. Shahbazi, P. V. Almeida, E. Mäkilä, A. Correia, M. P. A. Ferreira, M. Kaasalainen, J. Salonen, J. Hirvonen, H. A. Santos, Macromol. Rapid Commun. 2014, n/a; b) C. M. Koo, B. H. Jeon, I. J. Chung, J. Colloid Interface Sci. 2000, 227, 316. [9] a) D. Appelhans, H. Komber, M. A. Quadir, S. Richter, S. Schwarz, J. van der Vlist, A. Aigner, M. Muller, K. Loos, J. Seidel, K. F. Arndt, R. Haag, B. Voit, Biomacromolecules 2009, 10, 1114; b) M. S. Huh, S. Y. Lee, S. Park, S. Lee, H. Chung, S. Lee, Y. Choi, Y. K. Oh, J. H. Park, S. Y. Jeong, K. Choi, K. Kim, I. C. Kwon, J. Control Release 2010, 144, 134. [10] D. Liu, H. Zhang, B. Herranz, E. Mäkilä, V.-P. Lehto, J. Salonen, J. Hirvonen, H. A. Santos, Small 2014, doi: 10.1002/smll.201303740. [11] a) L. M. Bimbo, M. Sarparanta, H. A. Santos, A. J. Airaksinen, E. Makila, T. Laaksonen, L. Peltonen, V. P. Lehto, J. Hirvonen, J. Salonen, ACS Nano 2010, 4, 3023; b) L. M. Bimbo, E. Makila, J. Raula, T. Laaksonen, P. Laaksonen, K. Strommer, E. I. Kauppinen, J. Salonen, M. B. Linder, J. Hirvonen, H. A. Santos, Biomaterials 2011, 32, 9089; c) M. Kilpelainen, J. Riikonen, M. A. Vlasova, A. Huotari, V. P. Lehto, J. Salonen, K. H. Herzig, K. Jarvinen,
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
www.advmat.de www.MaterialsViews.com
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
COMMUNICATION
J. Control. Release 2009, 137, 166; d) M. P. Sarparanta, L. M. Bimbo, E. M. Makila, J. J. Salonen, P. H. Laaksonen, A. M. K. Helariutta, M. B. Linder, J. T. Hirvonen, T. J. Laaksonen, H. A. Santos, A. J. Airaksinen, Biomaterials 2012, 33, 3353. [12] L. Liu, J. P. Yang, X. J. Ju, R. Xie, L. Yang, B. Liang, L. Y. Chu, J. Colloid Interface Sci. 2009, 336, 100. [13] T. Irie, M. Tsujii, S. Tsuji, T. Yoshio, S. Ishii, S. Shinzaki, S. Egawa, Y. Kakiuchi, T. Nishida, M. Yasumaru, H. Iijima, H. Murata, T. Takehara, S. Kawano, N. Hayashi, Int. J. Cancer 2007, 121, 878.
[14] R. Chen, Y. Qian, R. Li, Q. Zhang, D. Liu, M. Wang, Q. Xu, Yakugaku Zasshi. 2010, 130, 419. [15] a) H. Zhang, M. A. Shahbazi, E. M. Makila, T. H. da Silva, R. L. Reis, J. J. Salonen, J. T. Hirvonen, H. A. Santos, Biomaterials 2013, 34, 9210; b) L. M. Bimbo, E. Makila, T. Laaksonen, V. P. Lehto, J. Salonen, J. Hirvonen, H. A. Santos, Biomaterials 2011, 32, 2625. [16] D. Liu, E. Mäkilä, H. Zhang, B. Herranz, M. Kaasalainen, P. Kinnari, J. Salonen, J. Hirvonen, H. A. Santos, Adv. Funct. Mater. 2013, 23, 1893.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
7
COMMUNICATION
Fabrication of a Multifunctional Nano-in-micro Drug Delivery Platform by Microfluidic Templated Encapsulation of Porous Silicon in Polymer Matrix Hongbo Zhang,* Dongfei Liu, Mohammad-Ali Shahbazi, Ermei Mäkilä, Bárbara Herranz-Blanco, Jarno Salonen, Jouni Hirvonen, and Hélder A. Santos* Orally administrated targeted drug delivery systems (TDDS) to the desired location in the gastrointestinal (GI) tract are highly demanded for the treatment of local diseases, such as colorectal cancer.[1] However, due to the harsh conditions and manifold barriers of the GI tract, many drugs are hindered from reaching the targeted site.[2] Uniform particle size is critical for these drug delivery systems, since it might significantly affect the biological interactions of the payloads in vivo.[3] An ideal advanced TDDS should efficiently protect the drugs in the varying GI conditions and precisely release the drugs at the targeted site. TDDS should have preferably high drug loading capacity, with the possibility to load multiple drugs with different physicochemical properties, especially for the treatment of complex diseases like cancers. Moreover, mucoadhesive properties of the TDDS are highly preferable to prolong the retention time of the drugs at the site of absorption, thus improving the therapeutic efficacy and bioavailability. Droplet-based microfluidics is a promising technique that can be applied to manipulate nanoliter volumes of immiscible carrier phases in the networks of micron scale channels and precisely dispersing these solutions into equally sized microdroplets; subsequently, the droplets can be solidified to form monodisperse microparticles.[3,4] The advantages of the microfludic technique are the possibility of fabricating monodisperse particulate structures, as well as the flexibility in material selection with an encapsulation efficiency of almost 100%.[3,4] Porous silicon (PSi) has been intensively investigated in drug delivery applications.[5] This material has unique properties such as high surface-to-volume ratio, high drug-loading capacity and the potential to deliver chemical drugs with multiple structures, proteins, siRNA, and viruses.[5d,6] Moreover, Dr. H. B. Zhang, D. F. Liu,[+] M. A. Shahbazi,[+] E. Mäkilä, B. Herranz-Blanco, Prof. J. Hirvonen, Prof. H. A. Santos Division of Pharmaceutical Chemistry and Technology Faculty of Pharmacy University of Helsinki Helsinki, FI-00014, Finland E-mail: [email protected]; [email protected] E. Mäkilä, Prof. J. Salonen Laboratory of Industrial Physics Department of Physics University of Turku Turku, FI-20014, Finland [+]These authors contributed equally to this work
DOI: 10.1002/adma.201400953
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
PSi has not only a good biocompatibility and biodegradability, but also the PSi surface can be chemically modified to present reactive groups for subsequent conjugation with different polymers, fluorescent probes, contrast agents, and biologically active targeting moieties.[5d,7] All these advantages make the PSi particles an ideal choice for drug delivery and biomedical applications. Typical mucoadhesive polymers have active groups capable to form strong hydrogen bonds, together with appropriate high molecular weight and density for cross-linking.[2] Among the mucoadhesive polymers, poly(methyl vinyl ether-co-maleic acid) (PMVEMA) polymer was selected for this study due to its overall favorable properties.[8] Herein, we conjugated the PMVEMA with carboxylic acid modified thermally hydrocarbonized porous silicon nanoparticles (PSi NPs) using polyethyleneimine (PEI) as a linker polymer. PEI was introduced due to its high density of amine groups to facilitate the conjugation of PMVEMA. In addition, the positively charged amine groups of PEI can also aid in enhancing the cellular interactions of the produced particles.[9] Subsequently, the microfluidic technique was applied to fabricate multistage TDDSs by encapsulating the PSi-PEI-PMVEMA inside a pH-responsive hydroxypropylmethylcellulose acetate succinate based polymer (ASHF).[10] The PSi NPs (average particle size of 151.1 ± 4.0 nm and zeta-potential of −36.0 ± 0.6 mV) were fabricated using electrochemical anodization, followed by thermal hydrocarbonization, surface modification with undecylenic acid and milling as described elsewhere.[11] PEI and PMVEMA were conjugated to the PSi NPs via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS) chemical reaction to form PSi-PEI-PMVEMA (average particle size of 366.7 ± 3.0 nm and zeta-potential of −34.3 ± 2.0 mV) (Figure 1a).[8a] The success of the polymer conjugation was evaluated by Fourier transform infrared spectroscopy (FTIR), where the characteristic peaks from the PSi, PEI, and PMVEMA were also detected from the PSi-PEI-PMVEMA spectrum (Figure S1a). The PSi-PEI-PMVEMA NPs were subsequently encapsulated in ASHF to form the nano-in-micro composites, PSiPEI-PMVEMA@ASHF (average particle size of ca. 30 µm), using a flow focusing microfluidic chip for producing an oilin-water (O/W) emulsion (Figure 1a). Ethyl acetate (EA) was used as a non-toxic organic solvent, which has fast diffusion rate to water due to its high solubility.[12] These properties are beneficial for producing particles for drug delivery applications with good stability and high encapsulation efficiency.[12] Thus,
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
1
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
Figure 1. (a) Schematic illustration of the fabrication process of the PSi-PEI-PMVEMA@ASHF composites. The PSi-PEI-PMVEMA NPs were prepared by conjugation with the PEI and PMVEMA polymers. Then, the PSi-PEI-PMVEMA NPs were encapsulated in the pH-responsive ASHF polymer with microfluidics. The 5-FU@PSi-PEI-PMVEMA NPs were dispersed in the inner fluid together with ASHF and CEL in EA. The outer continuous fluid contained 2% of P-407. (b) The monodispersity of the PSi-PEI-PMVEMA NPs of the fabricated PSi-PEI-PMVEMA@ASHF composite were studied by confocal microscope and SEM. The PSi-PEI-PMVEMA NPs were conjugated with FTIC and the ASHF layer was labeled with TRITC. The confocal images showed (from left to right) the FITC, TRITC and the overlay channels. In addition, the surface of the particles was monitored by SEM. (c) Dissolution behavior of the PSi-PEI-PMVEMA@ASHF composite at pH 7.4 monitored from 2 to 30 min by confocal microscopy; all images presented are the overlay channels.
PSi-PEI-PMVEMA along with the ASHF in EA solution was used as the inner fluid. A non-toxic amphiphilic Poloxamer 407 (P-407) was dissolved at pH 5.5 and was used as the outer fluid due to its ability to stabilize the formed droplets. In addition, the hydrophilic fluorouracil (5-FU) and the hydrophobic drug celecoxib (CEL), which act synergistically for colon cancer,[13] were loaded inside the composite. Due to the distinct physicochemical properties of both drugs, it is very challenging to first encapsulate and subsequently release the drugs from one single formulation. CEL has good solubility in EA and it was directly dissolved in this solvent. PSi-PEI-PMVEMA NPs were introduced within the ASHF polymer matrix to improve the drug loading capacity of the produced composite. By loading the 5-FU inside the PSi-PEI-PMVEMA NPs and then encapsulating the drug-loaded NPs inside the ASHF polymer, we 2
wileyonlinelibrary.com
were able to increase the loading degree of 5-FU from 0.5% to 7.1% (w/w). Moreover, the loading ratios of CEL to 5-FU could be freely adjusted. Herein, we tailored the drug loading ratio to approximately 1:1 by adjusting the loading degree of CEL to 6.9% (w/w). The success of the drugs encapsulation was confirmed by FTIR (Figure S1b). The flow rates in the two channels of the microfluidic chip were optimized in order to obtain particles with the desired size and size distribution. The inner fluid was fixed at 1500 µL/h and the outer fluid was adjusted to the flow at a rate of 15000 µL/h. Thus, we were able to obtain a particle size of ca. 50 µm by jetting regime (Figure S2). By decreasing the flow rate of the outer fluid from 15000 µL/h to 6000 µL/h, the flow changed from jetting to dripping regime (Figure S2), and the particle size decreased to ca. 30 µm (Figure 1b) with improved
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
www.advmat.de www.MaterialsViews.com
COMMUNICATION Figure 2. (A) Dissolution behavior of the PSi-PEI-PMVEMA@ASHF composite at different pH-values. The SEM images show the apparent morphology and surface properties of the particles after 2 h at different pH-conditions. By incubating at pH 7.4 for 2 h, the spherical composite totally dissolved and in the image we can only observe a polymer matrix. (B) Drug release profiles of 5-FU and CEL from the PSi-PEI-PMVEMA@ASHF composites in a series of pH buffers at 37 °C. The mixture of free 5-FU and CEL (ratio 1:1) was used as control. Data represent mean ± s.d. (n = 3).
(narrow) size distribution (Figure S2). These results are in good agreement with the previous findings that a dripping regime is more favorable in producing monodisperse emulsions.[14] The drops were collected in 2% P-407 at pH 5.5 (same as the outer fluid) and the droplets were solidified by diffusion of the EA to the outer aqueous phase. Due to the high water solubility of EA, fast solidification process could be achieved and can be further accelerated by frequently changing the outer aqueous solution, which will significantly improve also the monodispersity of the fabricated particles.[12] After drying in vacuum oven, the particles were collected as powders, and remained stable during and after long-term storage (results not shown). Since no toxic solvents were used during the production, the obtained particles can be administered orally. The monodispersity of the produced PSi-PEI-PMVEMA@ ASHF composites was evaluated by confocal microscopy, particularly the size distribution of the composites and the PSi-PEIPMVEMA NPs distribution within the composites. To enable the visualization, the PSi-PEI-PMVEMA NPs were conjugated with fluorescein isothiocyanate (FITC, green) and the outer ASHF polymer layer was labeled with tetramethylrhodamine (TRITC, red). The confocal images indicated that the PSi-PEIPMVEMA NPs were very well dispersed within the PSi-PEIPMVEMA@ASHF composites (Figure 1b). In addition to the confocal imaging, scanning eletron microscopy (SEM) images showed that the composites had smooth surfaces and spherical structures (Figure 1b). Furthermore, the efficient encapsulation of the PSi-PEI-PMVEMA NPs within the ASHF polymer droplets can be observed in Figure S2 and in Video S1. The dissolution behavior of the PSi-PEI-PMVEMA@ASHF composites at pH 7.4 was observed by confocal microscopy (Figure 1c). The composite’s outer layer, ASHF, started to dissolve immediately and the PSi-PEI-PMVEMA NPs (green) were released from the composites after 2 min of incubation at pH 7.4 (Figure 1c). The composites were fully collapsed after
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
30 min (Figure 1c). In addition, the pH-responsive properties of the composites were monitored with SEM by incubating them at pH 1.2, 5.5 and 6.5 for 2 h, and at pH 7.4 for 5 min and 2 h (Figure 2a). The composites showed clear pH-responsive properties, presenting a stable structure at pH ≤ 6.5 and dissolving fast at pH 7.4 (Figure 2a). The surface topology of the composites before and after dissolution is shown in Figure S3. To further evaluate the pH-responsive properties of the PSiPEI-PMVEMA@ASHF composites, the drug release kinetics of 5-FU and CEL from the composites were studied by incubation in a series of pH buffers, starting from pH 1.2 to pH 5.5 and 6.5 for a time period of 2 h for each pH-value, and then at pH 7.4 for 8 h in order to mimic the intestinal and colonic pH and transit time of the GI tract. Since CEL is insoluble in water, 5% of P-407 was added into the release buffers. The results showed that 5-FU and CEL were almost completely dissolved after 1 and 2 h at pH 1.2. The composites successfully encapsulated 5-FU and CEL within the ASHF polymer layer, which showed no drug release at pH ≤ 6.5 (Figure 2b). By contrast, at pH 7.4, after the ASHF polymer layer was dissolved, both 5-FU and CEL were completely released within 2 h (Figure 2b). The results indicated that the drug release from the composite was clearly controlled by the outer ASHF polymeric layer. Owing to the advantages of the microfluidic technique, by changing the ASHF polymer with different pH/temperature/enzyme responsive polymers, our platform has the potential to be used in different kinds of therapeutic applications. In addition to the efficient and controlled drug delivery properties of the aforementioned composites, the TDDS should also overcome the mucus layer of the intestine, which plays an important role in protecting the epithelial cells in the GI tract, and thus, creating a barrier for drug absorption.[2] In order to enhance the therapeutical efficiency of the drugs, mucoadhesive drug formulations are often needed.[2] To achieve this, herein the PSi NPs were conjugated with PEI and PMVEMA,
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
3
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
and the effect of the mucoadhesivity was tested in Caco-2/HT-29 co-culture monolayers. Different particles were incubated with Caco-2/HT-29 monolayers for 6 h at 37°C, and at pH 7.4. In all samples, the morphology of the Caco-2/HT-29 monolayers was not affected during incubation (Figure 3a), which indicates that these particles are not toxic to Caco-2/HT-29 monolayers under the experimental conditions tested. Clear differences on the mucoadhesion between PSi and PSi-PEI-PMVEMA NPs were observed; PSi-PEI-PMVEMA NPs were observed on top of the microvilli and almost no PSi NPs were detected (Figure 3a). This proved the fact that the mucoadhesion properties of PSiPEI-PMVEMA NPs were due to the surface modification with PEI and PMVEMA. In addition, the ASHF encapsulation did not affect the mucoadhesion of PSi-PEI-PMVEMA NPs since the PSi-PEI-PMVEMA NPs released from the composites at pH 7.4 also adhered to the surface of the Caco-2/HT-29 monolayers (Figure 3a).
The mucoadhesion and cellular interactions of the PSi-PEIPMVEMA@ASHF composites were further investigated in the presence of drugs. The 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composite was incubated with Caco-2/HT-29 monolayers at the pH-values of 6.5 and 7.4. At pH 6.5, no PSi-PEI-PMVEMA NPs were observed in the TEM images. In addition, the morphology of the Caco-2/HT-29 monolayers was not affected (no release of drugs, hence no toxicity to the cells) (Figure 3b), which further proved the successful encapsulation of the payloads (PSiPEI-PMVEMA NPs and the drugs) within the composite. At pH 7.4, the ASHF polymer layer was dissolved and the drugs and PSi-PEI-PMVEMA NPs were released (Figure 3b). The PSiPEI-PMVEMA NPs adhered to the Caco-2/HT-29 monolayers, and few PSi-PEI-PMVEMA NPs were taken up and transported to the basolateral side of the Caco-2/HT-29 monolayers (Figure 3b). Since PEI has the ability to improve the cellular internalization and endosomal escape,[9] the cellular uptake and
Figure 3. (a) Mucoadhesion properties of the PSi and PSi-PEI-PMVEMA NPs, as well as PSi-PEI-PMVEMA NPs released from the PSi-PEI-PMVEMA@ ASHF composites at pH 7.4. Particles at a concentration of 100 µg/mL were incubated with Caco-2/HT-29 monolayers at pH 7.4 and 37 °C for 6 h. (b) Cellular interaction of the 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites with Caco-2/HT-29 monolayers at the pH-values of 6.5 and 7.4. All scale bars in (a) and (b) are 500 nm. (c) Drug permeation profiles across Caco-2/HT-29 monolayers. The initial drug concentration in apical side was 100 µg/mL for 5-FU and CEL. The 5-FU&CEL@PSi-PEI-PMVEMA-ASHF concentrations were calculated similarly to the pure drugs based on the drug loading degree. The cells were exposed to the bare drugs or drug-loaded PSi-PEI-PMVEMA@ASHF composites at pH 7.4 and 37 °C for 3 h. Data represent mean ± s.d. (n = 3). The level of significances between the permeability of pure drugs and 5-FU&CEL@PSi-PEI-PMVEMA-ASHF were set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001.
4
wileyonlinelibrary.com
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
www.advmat.de www.MaterialsViews.com
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
COMMUNICATION
drug transport may have been due to the presence of PEI on the surface of the PSi NPs, and disruption of the cell monolayer when in contact with the drugs (no cell morphology disruption and particle uptake was observed in the absence of drugs, as shown in Figure 3a). The distinct different behaviour of the loaded drugs in the composites at the pH-values of 6.5 and 7.4 showed that the composites had the ability to protect the payloads from premature release and ensured successful control of drug release. These properties will significantly decrease the adverse drug’s effects and enhance the therapeutic efficiency of the drug(s). The effect of the PSi-PEI-PMVEMA@ASHF composite on the drug permeation across Caco-2/HT-29 cell monolayers was another important parameter to be evaluated for the oral drug delivery systems.[15] Herein, we also investigated the permeation of 5-FU and CEL across Caco-2/HT-29 cell monolayers before and after loading them into the composites. The results indicated that the permeability of both 5-FU and CEL were enhanced by loading them into the composites (Figure 3c). The apparent permeability coefficients (Papp) of 5-FU and CEL were increased from 1.30 to 1.62 (10−5 cm/s) and 0.67 to 0.91 (10−5 cm/s), respectively. In addition, the change of the transepithelial electrical resistance (TEER) of Caco-2/HT-29 monolayers during the drug permeation experiments was also monitored, which may help in predicting the paracellular permeability of the drugs.[15a] The results showed that the mixture of the pure drugs had a clear effect on reducing the TEER-values of the Caco-2/HT-29 monolayers (Figure S4), probably induced by the cytotoxicity of the drugs. This is in good agreement with the TEM images, which showed that in the presence of drugs the PSi-PEI-PMVEMA NPs were taken up and transported to the basoleteral sites of the Caco-2/HT-29 monolayers (Figure 3b). Interestingly, we observed more extensive TEER reduction of the 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites. This is because after the dissolution of the ASHF polymer layer, the PSi-PEI-PMVEMA NPs increased the mucoadhesivity and enhanced the release of the drugs in the vicinity of the monolayers, thus enhancing also the drug permeability. In order to assess the safety of the PSi-PEI-PMVEMA@ ASHF composites, we also tested the cell viabilities of Caco-2 and HT-29 cells at the pH-values 6.5 and 7.4 (Figure S5) after incubation with the different particles. At pH 6.5, the composites showed no significant toxicity at all the concentrations tested (25 to 250 µg/mL), whereas both PSi and PSi-PEIPMVEMA NPs showed some toxicity to the cells, especially at higher concentrations (Figures S5a and S5c). At pH 7.4, the ASHF polymer layer of the composites was dissolved and the PSi-PEI-PMVEMA NPs were exposed to the cells. As a result, only a slight difference on the cell viability was observed between the PSi-PEI-PMVEMA NPs and the composites (Figure S5b and 5d). Moreover, both Caco-2 and HT-29 cells behaved similarly, which showed that the toxicity was not cell type dependent. The different behaviour of the composites on the cell viability at different pH-values can be attributed to the successful encapsulation of the PSi-PEI-PMVEMA NPs and the pH-responsive of the composites. The impact of the 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites on the cell proliferation of Caco-2 and HT-29 cells at the pH-values 6.5 and pH 7.4 was also evaluated. At both
pH-values, the pure drug combinations significantly inhibited cell proliferation (Figure 4). The composites had no significant effect on the cell proliferation at pH 6.5, which was due to the absence of drug release (Figure 4). However, the composites remarkably inhibited the cell proliferations of both cell types at pH 7.4, and the inhibition effect was even stronger than with the mixture of both pure drugs. This phenomenon could be ascribed to the high mucoadhesive property of the PSi-PEIPMVEMA NPs to the cells’ surface and to the local accumulation of the drugs in the vicinity of the cells. The proliferation results are also in good agreement with both the drug release experiments (Figure 2b) and the TEM images of the composite’s mucoadhesion properties (Figure 3b). In conclusion, we have developed here a novel PSi-PEIPMVEMA@ASHF carrier system for TDDS by microfluidics. The composite showed well-defined spherical structure, narrow size distribution, and smooth surface. The particles can be dried as free-blowing powders and re-dispersed without damaging their structure. The fabricated composites showed promising properties for targeted drug delivery, including the capacity of loading multidrugs with different physicochemical properties (with precisely adjusting the drug loading ratios
Figure 4. Cell proliferation profiles of (a) Caco-2 and (b) HT-29 cells treated with series concentrations of mixtures of 5-FU and CEL, and the corresponding concentrations of 5-FU&CEL@PSi-PEI-PMVEMA-ASHF (based on drug loading degree) at the pH-values 6.5 and 7.4. Lines A and C are 5-FU and CEL drug mixtures at the pH-values 6.5 and pH 7.4; lines B and D are 5-FU&CEL@PSi-PEI-PMVEMA-ASHF composites at the pH-values 6.5 and pH 7.4. The cells were incubated at 37 °C for 24 h in the presence of 5% P-407. Data represent mean ± s.d. (n = 3). The level of significances between the inhibition curve of the pure drugs and the drug-loaded composites under the same pH-value are set at probabilities of *p < 0.05, **p < 0.01 and ***p < 0.001.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
5
www.advmat.de
COMMUNICATION
www.MaterialsViews.com
among the different drugs); successful encapsulation of the payloads within a pH-responsive matrix; simultaneous release of multidrugs; and controllable inhibition of the cancer cell proliferation in response to the pH changes. This TDDS was extremely stable at pH ≤ 6.5 and completely protected the payloads from premature release, but allowed fast release at pH 7.4. Furthermore, the released PSi-PEI-PMVEMA NPs showed strong mucoadhesion properties, therefore enabling the nanoin-micro composites to enhance the drug permeability and strengthen the inhibition effect on cell proliferation at pH 7.4. Owing to the properties of the composites and the flexibility of the microfluidic technique, the developed composites can be further extended as a platform for a variety of therapeutical applications.
Experimental Section The fabrication of the PSi NPs is described elsewhere.[11] PEI and PMVEMA were conjugated to PSi NPs via EDC/NHS chemical reaction to form PSi-PEI-PMVEMA (Figure 1). The success of the conjugation and characterization of the drug loading was studied by FTIR (Vertex 70, Bruker, USA), using a horizontal attenuated total reflectance accessory (MIRacle, PIKE Technologies, USA). PSi-PEI-PMVEMA was subsequently encapsulated with ASHF, using a flow focusing microfluidic chip to produce an oil-in-water (O/W) emulsion. 5-FU and CEL were chosen as model drugs; 5-FU was loaded into the PSi-PEI-PMVEMA NPs by using an immersing method as described elsewhere,[15a,16] and CEL was dissolved in the inner fluid in EA. The inner fluid contained 5-FU loaded PSi-PEI-PMVEMA NPs, ASHF and CEL in EA and the outer fluid was 2% P-407, pH 5.5 (Figure 1). The monodisperse distribution of PSi-PEI-PMVEMA NPs within the ASHF polymeric layer was studied by an inverted confocal fluorescence microscope (Leica SP5 II HCS A, Germany) and the surface morphology of the PSi-PEI-PMVEMA@ASHF composites was monitored by a Quanta 250 SEM (FEG, USA). The pH-responsive properties of the PSi-PEI-PMVEMA@ASHF composites were studied by SEM imaging after incubating the composites with different pH buffers (1.2, 5.5, 6.5 and 7.4). In addition, the dissolution of the composites at pH 7.4 was recorded with confocal microscope between 2 to 30 min. The drug loading degree and release kinetics were analyzed by HPLC (details can be found in the Supporting Information). The drug release was studied under a series of pH buffers (pH 1.2, 5.5, 6.5 and 7.4) with gradient. The mucoadhesion property of the PSi and PSi-PEI-PMVEMA NPs was studied by incubating different particles with Caco-2/HT-29 monolayers and then imaging them by TEM (Jeol JEM1400, Jeol Ltd., Japan). The drug permeability across the cell monolayers and TEER-values were also measured (see details in Supporting Information). Last, we studied the cell viability and the cell proliferation in Caco-2 and HT-29 cells at the pH-values 6.5 and 7.4 with an ATPbased CellTiter-Glo assay (Promega Corporation, USA). Further detailed experimental procedures are provided in the Supporting Information.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements The financial support from the Academy of Finland (decision numbers 252215 and 256394), the University of Helsinki Funds, Biocentrum
6
wileyonlinelibrary.com
Helsinki, and the European Research Council under the European Union’s Seventh Framework Programme (FP/2007–2013, Grant No. 310892) are highly acknowledged. HARKE Pharma (Germany) is acknowledged for kindly providing the ASHF pH-responsive polymer (Shin-Etsu AQOAT). Dr. H. Zhang acknowledges the Finnish Cultural Foundation for a Post-doc Pooli grant. Received: February 28, 2014 Revised: March 23, 2014 Published online:
[1] Y. S. Krishnaiah, M. A. Khan, Pharm. Dev. Technol. 2012, 17, 521. [2] M. A. Shahbazi, H. A. Santos, Curr. Drug Metab. 2013, 14, 28. [3] W. J. Duncanson, T. Lin, A. R. Abate, S. Seiffert, R. K. Shah, D. A. Weitz, Lab Chip 2012, 12, 2135. [4] a) S. Yang, F. Guo, B. Kiraly, X. Mao, M. Lu, K. W. Leong, T. J. Huang, Lab Chip 2012, 12, 2097; b) D. Liu, B. Herranz-Blanco, E. Mäkilä, L. R. Arriaga, S. Mirza, D. A. Weitz, N. Sandler, J. Salonen, J. Hirvonen, H. A. Santos, ACS Appl. Mater. Inter. 2013, 5, 12127; c) B. Herranz-Blanco, L. R. Arriaga, E. Makila, A. Correia, N. Shrestha, S. Mirza, D. A. Weitz, J. Salonen, J. Hirvonen, H. A. Santos, Lab Chip 2014. [5] a) A. Parodi, N. Quattrocchi, A. L. van de Ven, C. Chiappini, M. Evangelopoulos, J. O. Martinez, B. S. Brown, S. Z. Khaled, I. K. Yazdi, M. V. Enzo, L. Isenhart, M. Ferrari, E. Tasciotti, Nat. Nanotechnol. 2013, 8, 61; b) E. Tasciotti, X. W. Liu, R. Bhavane, K. Plant, A. D. Leonard, B. K. Price, M. M. C. Cheng, P. Decuzzi, J. M. Tour, F. Robertson, M. Ferrari, Nat. Nanotechnol. 2008, 3, 151; c) J. H. Park, L. Gu, G. von Maltzahn, E. Ruoslahti, S. N. Bhatia, M. J. Sailor, Nat. Mater. 2009, 8, 331; d) H. A. Santos, L. M. Bimbo, V. P. Lehto, A. J. Airaksinen, J. Salonen, J. Hirvonen, Curr. Drug Disc. Technol. 2011, 8, 228. [6] a) L. M. Bimbo, O. V. Denisova, E. Mäkilä, M. Kaasalainen, J. K. De Brabander, J. Hirvonen, J. Salonen, L. Kakkola, D. Kainov, H. A. Santos, ACS Nano 2013, 7, 6884; b) D. Liu, L. M. Bimbo, E. Makila, F. Villanova, M. Kaasalainen, B. Herranz-Blanco, C. M. Caramella, V. P. Lehto, J. Salonen, K. H. Herzig, J. Hirvonen, H. A. Santos, J. Control. Release 2013, 170, 268. [7] a) C.-F. Wang, E. M. Mäkilä, M. H. Kaasalainen, D. Liu, M. P. Sarparanta, A. J. Airaksinen, J. J. Salonen, J. T. Hirvonen, H. A. Santos, Biomaterials 2014, 35, 1257; b) L. M. Bimbo, M. Sarparanta, E. Makila, T. Laaksonen, P. Laaksonen, J. Salonen, M. B. Linder, J. Hirvonen, A. J. Airaksinen, H. A. Santos, Nanoscale 2012, 4, 3184. [8] a) M.-A. Shahbazi, P. V. Almeida, E. Mäkilä, A. Correia, M. P. A. Ferreira, M. Kaasalainen, J. Salonen, J. Hirvonen, H. A. Santos, Macromol. Rapid Commun. 2014, n/a; b) C. M. Koo, B. H. Jeon, I. J. Chung, J. Colloid Interface Sci. 2000, 227, 316. [9] a) D. Appelhans, H. Komber, M. A. Quadir, S. Richter, S. Schwarz, J. van der Vlist, A. Aigner, M. Muller, K. Loos, J. Seidel, K. F. Arndt, R. Haag, B. Voit, Biomacromolecules 2009, 10, 1114; b) M. S. Huh, S. Y. Lee, S. Park, S. Lee, H. Chung, S. Lee, Y. Choi, Y. K. Oh, J. H. Park, S. Y. Jeong, K. Choi, K. Kim, I. C. Kwon, J. Control Release 2010, 144, 134. [10] D. Liu, H. Zhang, B. Herranz, E. Mäkilä, V.-P. Lehto, J. Salonen, J. Hirvonen, H. A. Santos, Small 2014, doi: 10.1002/smll.201303740. [11] a) L. M. Bimbo, M. Sarparanta, H. A. Santos, A. J. Airaksinen, E. Makila, T. Laaksonen, L. Peltonen, V. P. Lehto, J. Hirvonen, J. Salonen, ACS Nano 2010, 4, 3023; b) L. M. Bimbo, E. Makila, J. Raula, T. Laaksonen, P. Laaksonen, K. Strommer, E. I. Kauppinen, J. Salonen, M. B. Linder, J. Hirvonen, H. A. Santos, Biomaterials 2011, 32, 9089; c) M. Kilpelainen, J. Riikonen, M. A. Vlasova, A. Huotari, V. P. Lehto, J. Salonen, K. H. Herzig, K. Jarvinen,
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
www.advmat.de www.MaterialsViews.com
Adv. Mater. 2014, DOI: 10.1002/adma.201400953
COMMUNICATION
J. Control. Release 2009, 137, 166; d) M. P. Sarparanta, L. M. Bimbo, E. M. Makila, J. J. Salonen, P. H. Laaksonen, A. M. K. Helariutta, M. B. Linder, J. T. Hirvonen, T. J. Laaksonen, H. A. Santos, A. J. Airaksinen, Biomaterials 2012, 33, 3353. [12] L. Liu, J. P. Yang, X. J. Ju, R. Xie, L. Yang, B. Liang, L. Y. Chu, J. Colloid Interface Sci. 2009, 336, 100. [13] T. Irie, M. Tsujii, S. Tsuji, T. Yoshio, S. Ishii, S. Shinzaki, S. Egawa, Y. Kakiuchi, T. Nishida, M. Yasumaru, H. Iijima, H. Murata, T. Takehara, S. Kawano, N. Hayashi, Int. J. Cancer 2007, 121, 878.
[14] R. Chen, Y. Qian, R. Li, Q. Zhang, D. Liu, M. Wang, Q. Xu, Yakugaku Zasshi. 2010, 130, 419. [15] a) H. Zhang, M. A. Shahbazi, E. M. Makila, T. H. da Silva, R. L. Reis, J. J. Salonen, J. T. Hirvonen, H. A. Santos, Biomaterials 2013, 34, 9210; b) L. M. Bimbo, E. Makila, T. Laaksonen, V. P. Lehto, J. Salonen, J. Hirvonen, H. A. Santos, Biomaterials 2011, 32, 2625. [16] D. Liu, E. Mäkilä, H. Zhang, B. Herranz, M. Kaasalainen, P. Kinnari, J. Salonen, J. Hirvonen, H. A. Santos, Adv. Funct. Mater. 2013, 23, 1893.
© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
wileyonlinelibrary.com
7
