Validation of a Janus role of methotrexate-based PEGylated chitosan nanoparticles in vitro
- Fanghong Luo†1, 2, 4,
- Yang Li†1, 2,
- Mengmeng Jia2,
- Fei Cui2,
- Hongjie Wu3,
- Fei Yu1,
- Jinyan Lin1, 2,
- Xiangrui Yang2,
- Zhenqing Hou2Email author and
- Qiqing Zhang5Email author
© Luo et al.; licensee Springer. 2014
Received: 17 June 2014
Accepted: 18 July 2014
Published: 23 July 2014
Recently, methotrexate (MTX) has been used to target to folate (FA) receptor-overexpressing cancer cells for targeted drug delivery. However, the systematic evaluation of MTX as a Janus-like agent has not been reported before. Here, we explored the validity of using MTX playing an early-phase cancer-specific targeting ligand cooperated with a late-phase therapeutic anticancer agent based on the PEGylated chitosan (CS) nanoparticles (NPs) as drug carriers. Some advantages of these nanoscaled drug delivery systems are as follows: (1) the NPs can ensure minimal premature release of MTX at off-target site to reduce the side effects to normal tissue; (2) MTX can function as a targeting ligand at target site prior to cellular uptake; and (3) once internalized by the target cell, the NPs can function as a prodrug formulation, releasing biologically active MTX inside the cells. The (MTX + PEG)-CS-NPs presented a sustained/proteases-mediated drug release. More importantly, compared with the PEG-CS-NPs and (FA + PEG)-CS-NPs, the (MTX + PEG)-CS-NPs showed a greater cellular uptake. Furthermore, the (MTX + PEG)-CS-NPs demonstrated a superior cytotoxicity compare to the free MTX. Our findings therefore validated that the MTX-loaded PEGylated CS-NPs can simultaneously target and treat FA receptor-overexpressing cancer cells.
KeywordsMethotrexate Chitosan Drug delivery system Tumor Nanoparticles
Nanotechnology is a rapidly advancing and key field of drug delivery. A great variety of nanoparticle (NP)-based therapeutic products have entered clinical development or been approved for clinical use . As an excellent biocompatible and biodegradable nanomaterial with low toxicity and immunogenicity, chitosan (CS)-based nanocarriers presented great advantages for drug, protein, and gene delivery in therapeutics [2–5]. However, most CS-based nanocarriers were easily sequestered by macrophages in the mononuclear phagocyte system (MPS) after intravenous administration. To avoid the rapid clearance of the CS-NPs during circulation, PEGylation can be used to improve the physiological stability, reduce the opsonization, and increase the possibility reaching the tumor by the enhanced permeation and retention (EPR) effect (40 to 400 nm) [6–8].
Despite these advantages of the passive targeting, the main obstacle encountered with the clinical use of the PEGylated CS-NPs is how to facilitate their internalization in the target cells while reducing the unintended side effects. One strategy is the further functionalization of the PEGylated CS-NPs with active targeting agents. For instance, some ligands or antibodies could specifically recognize the receptors or antigens on the surface of various cancer cells . Notably, the exploitation of folate (FA) receptor for targeted drug delivery has long been persued. FA receptors were overexpressed in a wide variety of cancer cells, including ovarian, lung, breast, kidney, and brain cancer cells, but its level is very low in normal cells [10, 11]. Previously, we synthesized the CS-NPs by the combination of ionic gelation and chemical cross-linking method and prepared the (FA + PEG)-CS-NPs by dual-conjugation with mPEG-SPA and FA ; the enhanced cellular uptake and tumor accumulation also inspired our motivation of adopting the CS-NPs as drug carriers to continue our studies for an extensively used anticancer drug methotrexate (MTX).
All chemical reagents were of analytical grade and used without further purification unless otherwise stated. Chitosan (CS, Mw = 70,000 Da, 95% degree of deacetylation) was purchased from Zhejiang Aoxing Biotechnology Co., Ltd. (Zhengjiang, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and crude proteases from bovine pancreas were purchased from Sigma Chemical Corp (St. Louis, MO, USA). Folate (FA) and methotrexate (MTX) were purchased from Bio Basic Inc. (Markham, Ontario, Canada). N-Succinimidyl ester of methoxypolyethylene glycol propionic acid (mPEG-SPA, Mw = 2,000 Da) was purchased from Jiaxing Biomatrix Inc. (Zhengjiang, China). A dialysis bag (Mw = 8,000 to 14,000 Da) was ordered from Greenbird Inc. (Shanghai, China). A Spectra/Por dialysis membrane (Mw = 6,000 to 8,000 Da) was purchased from Spectrum Laboratories (Rancho Domingues, CA, USA). Deionized (DI) water was used throughout. Fetal bovine serum (FBS) was purchased from Gibco Life Technologies (AG, Zug, Switzerland). Trypsin-EDTA (0.25%) and penicillin-streptomycin solution was from Invitrogen. All solvents used in this study were high-performance liquid chromatography (HPLC) grade. HeLa cells and MC 3 T3-E1 cells were provided by American Type Culture Collection (ATCC, Manassas, VA, USA).
Preparation of the (MTX + PEG)-CS-NPs
Firstly, the CS-NPs were prepared by the ionic gelation combined with chemical cross-linking method according to our previous work . Secondly, mPEG-SPA (50 mg) was added into the CS-NPs suspensions (5 mL, 10 mg/mL) accompanied by vigorous stirring for 4 h. The prepared PEG-CS-NPs were dialyzed against DI water to remove excess of mPEG-SPA using a dialysis bag (Mw = 8,000 to 14,000 Da) and lyophilized for 24 h. Lastly, MTX (5 mg), EDC (8 mg), and NHS (5 mg) were dissolved in 5 mL of PBS (pH = 7.4). The pH was adjusted to 6.0 by the addition of 0.2 M HCl. The mixture was allowed to react for 30 min and added dropwise to the PEG-CS-NPs suspension (5 mL, 10 mg/mL). The pH was adjusted to 8.0 with 0.2 M NaOH. The reaction was allowed to occur at room temperature for 48 h. Following MTX conjugation, the (MTX + PEG)-CS-NPs NPs were centrifuged at 20,000 rpm for 30 min at 4°C, washed with PBS/DI water, and lyophilized for 24 h. All of the supernatants were collected for further indirect calculation of the drug-loading content. The (FA + PEG)-CS-NPs were prepared by the same method.
Physicochemical characterization of (MTX + PEG)-CS-NPs
Fourier transform infrared spectroscopy (FTIR) spectrum analysis of (MTX + PEG)-CS-NPs was performed using a NicoletAVTAR36 FTIR Spectrometer (Thermo Scientific, Salt Lake City, UT, USA). For comparison, The CS-NPs, PEG, PEG-CS-NPs, and MTX were used as controls.
Average particle size and polydispersity index (PDI) were determined by dynamic light scattering (DLS) using a Malvern Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). Zeta potential was evaluated by electrophoretic light scattering (ELS) with Zetaplus (Brookhaven Instruments Corporation, Holtsville, NY, USA). Particle size was evaluated by intensity distribution. Atomic force microscopy (AFM) study was performed on a Nanoscope Multimode atomic force microscope (Veeco Instruments Inc., New York, USA). Transmission electron microscopy (TEM) image was obtained on a JEM 2100 transmission electron microscope (JEOL, Tokyo, Japan).
The amount of drug in the supernatant was assayed using a high-performance liquid chromatography (Waters Associates, Milford, MA, USA) system with the following conditions: stationary phase, Hypersill ODS column (250 mm × 4.6 mm, 5 μm); mobile phase, potassium dihydrogen phosphate buffer (pH 4.5)-acetonitrile (88:12); elution flow rate, 1 mL/min; and detection wavelength, 303 nm. The drug-loading content was calculated according to the previous report .
In vitro stability tests
PBS stability test against ionic strength and plasma stability test against protein adsorption were evaluated immediately after preparation and subsequently at regular intervals. Briefly, 5 mg of the lyophilized (MTX + PEG)-CS-NPs were suspended in PBS (pH 7.4) or 10% (v/v) plasma/heparin in PBS and stored at 37°C for 120 h. The particle size was determined at 0, 24, 48, 72, 96, and 120 h, respectively.
In vitro drug release
In vitro release of MTX from the (MTX + PEG)-CS-NPs was evaluated by a dialysis method. The lyophilized (MTX + PEG)-CS-NPs suspended in 10% plasma (with or without the presence of crude proteases) were added into a dialysis bag (Mw = 6,000 to 8,000 Da) and immersed into the release medium at 37°C with agitation. At the predesigned time points, 2 mL of the release medium was completely withdrawn and subsequently replaced with the same volume of fresh PBS. For comparison, in vitro release of the free MTX was evaluated as a control.
HeLa cells were cultured in FA-deficient Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. MC 3 T3-E1 cells were cultured in Minimum Essential Medium, Alpha Modified (α-MEM), under similar conditions. The two cell lines have different levels of FA receptor expression. In particular, HeLa cells (cancer cells) are FA receptor positive, and MC 3 T3-E1 cells (normal cells) are FA receptor negative. All of the cells were cultivated in a 5% CO2-humidified atmosphere at 37°C.
In vitro cellular uptake
To qualitatively investigate the cellular uptake of the PEG-CS-NPs, (FA + PEG)-CS-NPs or (MTX + PEG)-CS-NPs, fluorescein isothiocyanate (FITC) was conjugated to different formulations to prepare the FITC-PEG-CS-NPs, FITC-(FA + PEG)-CS-NPs or FITC-(MTX + PEG)-CS-NPs. HeLa cells were seeded at a density of 8 × 104 cells per well into 6-well plates with their specific cell culture medium. The cells were incubated at 37°C and 5% CO2 for 24 h. One hundred microliters of the FITC-PEG-CS-NPs, FITC-(FA + PEG)-CS-NPs, or FITC-(MTX + PEG)-CS-NPs was added to HeLa cells at the equivalent concentration of FITC and incubated further for 6 h. HeLa cells were washed with PBS and stained with Hoechst 33258. Then, HeLa cells were washed with PBS and fixed with 4% formaldehyde. The cells were observed using a Leica TCS SP5 laser confocal scanning microscopy (Leica Microsystems, Mannheim, Germany).
To quantitatively investigate the internalization of the FITC-labeled (MTX + PEG)-CS-NPs, (FA + PEG)-CS-NPs or PEG-CS-NPs, HeLa cells were incubated in 6-well plates at a density of 2 × 105 cells/mL and allowed to grow for 24 h. The FITC-(MTX + PEG)-CS-NPs, FITC-(FA + PEG)-CS-NPs, or FITC-PEG-CS-NPs at the equivalent concentration of FITC were then added to each well. After incubation for 4 h, the cells were washed with cold PBS twice, harvested by 0.25% (w/v) trypsin/0.03% (w/v) EDTA, centrifuged at 1,000 rpm for 5 min at 4°C and resuspended in PBS for the analysis by a Coulter EPICS XL Flow Cytometer (Beckman Coulter Inc., Brea, CA, USA).
In vitro cell viability studies
Cytotoxicity of the PEG-CS-NPs, (FA + PEG)-CS-NPs, (MTX + PEG)-CS-NPs, and free MTX were evaluated by MTT assay. HeLa cells (cancer cells) or MC 3 T3-E1 cells (normal cells) were seeded at a density of 3 × 103 cells per well into 96-well plates with their specific cell culture medium. The cells were incubated at 37°C in humidified atmosphere containing 5% CO2 for 24 h. The medium was then replaced with fresh medium, and different formulations were added to incubate with the cells. After 24 h of incubation, the medium was removed; each well was rinsed with PBS; and 20 μL of MTT solution was added followed by incubation for 4 h. Then, the metabolized product MTT formazan was dissolved by adding 200 μL of DMSO to each well. Finally, the plate was shaken for 20 min, and the absorbance of the formazan product was measured at 570 nm in a microplate reader (Bio-Rad, Model 680, Bio-Rad Laboratories, Richmond, CA, USA).
To further understand the mechanisms of in vitro cell viability studies, we investigated the subcellular localization using a laser confocal scanning microscopy. After the predesigned incubation times with the FITC-labeled (MTX + PEG)-CS-NPs, HeLa cells were washed with PBS and stained with LysoTracker Red following the manufacturer’s instructions. The cells were then washed with PBS, fixed with 4% formaldehyde for 15 min and observed by a laser confocal scanning microscopy.
Results and discussion
Preparation of the (MTX + PEG)-CS-NPs
Physicochemical characterization of the (MTX + PEG)-CS-NPs
Drug-loading content. CS-NPs possessing peripheral amino groups provided us great opportunities to easy surface biofunctionalization. In our study, the γ-carboxyl groups of MTX were conjugated to the residual amino groups of the PEGylated CS-NPs. The drug-loading content of the (MTX + PEG)-CS-NPs was calculated as 7.23 ± 0.11%. The simple conjugation chemistry and appropriate drug-loading content could favor the dual-acting role of Janus-like MTX.
In vitro stability tests
No significant variation of the particle size was observed in the (MTX + PEG)-CS-NPs even after incubation with PBS for a long period of time (Figure 4F). Notably, the CS-NPs (without PEGylation) could precipitate after 48 h in the presence of salts. It was implied that PEG could protect the (MTX + PEG)-CS-NP against ionic strength. No significant change of the particle size was also shown in the (MTX + PEG)-CS-NPs after incubation with 10% plasma for 120 h (Figure 4G). It should be inferred that PEG could reduce the plasma proteins adsorption, and more importantly, preserve the targeting potential of MTX. All of the results suggested that the (MTX + PEG)-CS-NPs were sufficiently stable to sustain physiological conditions for extended blood circulation.
In vitro drug release profiles
It was well established that the amide bonds could be selectively cleaved at acidic pH by proteases (also called proteolytic enzymes) overexpressed in the tumor cells [33–36]. To simulate the physiological environment of the acidic compartments of the tumor cells, the (MTX + PEG)-CS-NPs were incubated with 10% plasma containing crude proteases at pH 5.0. MTX was released at a constant rate up to 10 h, reaching the accumulated release amounts more than 30%, we believed that proteases exerted a significant promotion effect to control drug release. As is reported, several kinds of particle-bound MTX attached by an amide linkage have been shown to be sensitive to the protease-mediated cleavage in the acidic environments, and hence, the lysosomal proteases could be responsible for the release of MTX from the particles [19, 20, 37, 38]. Once the NPs were internalized by the target cells, the drug release could be significantly speeded up because of the long-lasting activity of proteases inside the cells, which can help to provide a sufficient intracellular level of MTX, and hence efficiently enhance the drug efficacy.
All of the results suggested that the covalent chemistry, preferring over physical adsorption, could be advantageous to preserve the targeting role of MTX. This could be of utmost importance, especially in vivo, where the avoidance of premature drug release and the untimely role change (from targeting to anticancer) of Janus-like MTX are pivotal.
In vitro cellular uptake
These quantitative results were consistent with those qualitative results, giving a further proof of high targeting efficacy of the (MTX + PEG)-CS-NPs to HeLa cells. The possible reason is that the integral binding avidity of the (MTX + PEG)-CS-NPs towards FA receptor presents a great advantage of targeting efficacy outperformed that of the (FA + PEG)-CS-NPs towards FA receptor. As mentioned above, MTX has a suboptimal affinity to FA receptor compared with FA and may be less efficient to target to FA receptor than FA. Nevertheless, it was reported that multivalent binding avidity can be kinetically limited if the binding affinity of an individual receptor-ligand pair is too tight [44, 45]. Well consistent with the above theoretical analysis, our result further suggested that the targeting specificity of the nanoscaled drug delivery systems for a particular cell type can be enhanced by the weaker binding affinity of each individual receptor-ligand pair. Indeed, the integral binding avidity plays a predominant role in the targeting efficacy; the higher integral binding avidity increases the targeting efficacy. Detailed in vivo targeting studies are necessary to further assess this possibility.
In vitro cell viability studies
The cytotoxicity of the (MTX + PEG)-CS-NPs (10 μg/mL) towards HeLa cells and MC 3 T3-E1 cells after 24 h of incubation was shown in Figure 8B. FA receptors were expressed at a high level on the surface of HeLa cells (cancer cells) but at a much lower level on MC 3 T3-E1 cells (normal cells). On the one hand, the cytotoxicity of the (MTX + PEG)-CS-NPs towards cancer cells was significantly higher compared to that of the free MTX. However, in the case of normal cells, the situation was opposite. On the other hand, the (MTX + PEG)-CS-NPs induced a marked cytotoxicity towards targeted cancer cells, but a slight cytotoxicity was observed for nontargeted normal cells, whereas the free drug affected both cell lines equally. The result indicated that the MTX modification played an important role in selectively enhanced cytotoxicity of the nanoscaled drug delivery systems . All of these results also suggested that MTX was not prematurely released from the (MTX + PEG)-CS-NPs outside of HeLa cell, but was preferentially released inside HeLa cell after the cellular uptake of the (MTX + PEG)-CS-NPs.
To investigate the intrinsical mechanisms of the cytotoxicity of the (MTX + PEG)-CS-NPs, we investigated the subcellular localization of the FITC-labeled (MTX + PEG)-CS-NPs in HeLa cells by laser scanning confocal microscopy. As shown in Figure 8C, the internalized (MTX + PEG)-CS-NPs were found initially to be localized in the lysosomes, as evidenced by the yellow spots in the merged image obtained from the images of the (MTX + PEG)-CS-NPs (green) and late endosomes/lysosomes (red). The result indicated that the (MTX + PEG)-CS-NPs were internalized via the endocytosis pathway into the late endosomes/lysosome . Indeed, after incubation for 4 h, some green fluorescent FITC-labeled (MTX + PEG)-CS-NPs were no longer located in the red fluorescent late endosomes/lysosomes, indicating the successful endo/lysosomal escape. In agreement with other reports [37, 48], these results combined with the results of in vitro drug release and cell viability studies further proved that MTX was released from the (MTX + PEG)-CS-NPs inside the cells by the intracellular protease-mediated selective cleavage of peptide bond. These findings were also in agreement with other reports in the literature  that CS possessed the activity to some extent to escape the endo/lysosome.
We presented the versatile, robust, and easy MTX-based PEGylated CS-NPs while validating MTX as a successful targeting ligand coordinated with a simple anticancer drug, and established the (MTX + PEG)-CS-NPs as a cocktail platform of specific targeting cooperated with enhanced anticancer activity. MTX was not prematurely released at off-target site but was intensively released at target site due to its sustained/protease-mediated drug release characteristic. To the best of our knowledge, the work for the first time explored the validation of Janus role of MTX based on the nanoscaled drug delivery system in vitro. Additionally, as MTX (a targeting ligand/a first drug) was introduced into one kind of drug carriers, one further advantage was that the drug delivery systems allowed the further introduction of a second ligand or a second drug for synergistic co-targeted delivery or synergistic co-delivery of drugs. Nevertheless, more details about in vivo targeting and anticancer investigations are indispensable to obtain a better understanding of the therapeutic effect of the (MTX + PEG)-CS-NPs, and relevant studies are in process.
Both authors FL and YL contributed equally and should be considered as co-first authors.
Fanghong Luo acknowledges the financial support by the Natural Science Foundation of Fujian Province of China (Grant No. 2013 J01384) and Science and Technology Foundation of Xiamen of China (Grant No. 3502Z20113012). Dr. Yuan Jiang is acknowledged for useful discussions and editing the manuscript.
- Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R: Nanocarriers as an emerging platform for cancer therapy. Nat Nanotechnol 2007, 2: 751–760.View ArticleGoogle Scholar
- Garcia-Fuentes M, Alonso MJ: Chitosan-based drug nanocarriers: where do we stand? J Control Release 2012, 161: 496–504.View ArticleGoogle Scholar
- Agnihotri SA, Mallikarjuna NN, Aminabhavi TM: Recent advances on chitosan-based micro- and nanoparticles in drug delivery. J Control Release 2004, 100: 5–28.View ArticleGoogle Scholar
- Amidi M, Mastrobattista E, Jiskoot W, Hennink WE: Chitosan-based delivery systems for protein therapeutics and antigens. Adv Drug Delivery Rev 2010, 62: 59–82.View ArticleGoogle Scholar
- Mao S, Sun W, Kissel T: Chitosan-based formulations for delivery of DNA and siRNA. Adv Drug Delivery Rev 2010, 62: 12–27.View ArticleGoogle Scholar
- Graf N, Bielenberg DR, Kolishetti N, Muus C, Banyard J, Farokhzad OC, Lippard SJ: αVβ3 integrin-targeted PLGA-PEG nanoparticles for enhanced anti-tumor efficacy of a Pt(IV) prodrug. ACS Nano 2012, 6: 4530–4539.View ArticleGoogle Scholar
- O’Neal DP, Hirsch LR, Halas NJ, Payne JD, West JL: Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles. Cancer Lett 2004, 209: 171–176.View ArticleGoogle Scholar
- Cui F, Li Y, Zhou S, Jia M, Yang X, Yu F, Ye S, Hou Z, Xie L: A comparative in vitro evaluation of self-assembled PTX-PLA and PTX-MPEG-PLA nanoparticles. Nanoscale Res Lett 2013, 8: 301.View ArticleGoogle Scholar
- Allen TM: Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer 2002, 2: 750–763.View ArticleGoogle Scholar
- Low PS, Henne WA, Doorneweerd DD: Discovery and development of folic-acid-based receptor targeting for imaging and therapy of cancer and inflammatory diseases. Acc Chem Res 2008, 41: 120–129.View ArticleGoogle Scholar
- Weitman SD, Lark RH, Coney LR, Fort DW, Frasca V, Zurawski VR Jr, Kamen BA: Distribution of the folate receptor GP38 in normal and malignant cell lines and tissues. Cancer Res 1992, 52: 3396–3401.Google Scholar
- Hou Z, Zhan C, Jiang Q, Hu Q, Li L, Chang D, Yang X, Wang Y, Li Y, Ye S, Xie L, Yi Y, Zhang Q: Both FA- and mPEG-conjugated chitosan nanoparticles for targeted cellular uptake and enhanced tumor tissue distribution. Nanoscale Res Lett 2011, 6: 563.View ArticleGoogle Scholar
- Rijnboutt S, Jansen G, Posthuma G, Hynes JB, Schornagel JH, Strous GJ: Endocytosis of GPI-linked membrane folate receptor-alpha. J Cell Biol 1996, 132: 35–47.View ArticleGoogle Scholar
- Mizusawa K, Takaoka Y, Hamachi I: Specific cell surface protein imaging by extended self-assembling fluorescent turn-on nanoprobes. J Am Chem Soc 2012, 134: 13386–13395.View ArticleGoogle Scholar
- Qiu A, Jansen M, Sakaris A, Min SH, Chattopadhyay S, Tsai E, Sandoval C, Zhao R, Akabas MH, Goldman ID: Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. Cell 2006, 127: 917–928.View ArticleGoogle Scholar
- Frei E, Jaffe N, Tattersall MHN, Pitman S, Parker L: New approaches to cancer chemotherapy with methotrexate. N Engl J Med 1975, 292: 846–851.View ArticleGoogle Scholar
- Matthews DA, Alden RA, Bolin JT, Freer ST, Hamlin R, Xuong N, Kraut J, Poe M, Williams M, Hoogsteen K: Dihydrofolate reductase: x-ray structure of the binary complex with methotrexate. Science 1977, 197: 452–455.View ArticleGoogle Scholar
- Roberts GC, Feeney J, Birdsall B, Charlton P, Young D: Methotrexate binding to dihydrofolate reductase. Nature 1980, 286: 309.View ArticleGoogle Scholar
- Kohler N, Sun C, Fichtenholtz A, Gunn J, Fang C, Zhang M: Methotrexate-immobilized poly(ethylene glycol) magnetic nanoparticles for MR imaging and drug delivery. Small 2006, 2: 785–792.View ArticleGoogle Scholar
- Rosenholm JM, Peuhu E, Bate-Eya LT, Eriksson JE, Sahlgren C, Linden M: Cancer-cell-specific induction of apoptosis using mesoporous silica nanoparticles as drug-delivery vectors. Small 2010, 6: 1234–1241.View ArticleGoogle Scholar
- Thomas TP, Huang B, Choi SK, Silpe JE, Kotlyar A, Desai AM, Zong H, Gam J, Joice M, Baker JR Jr: Polyvalent dendrimer-methotrexate as a folate receptor-targeted cancer therapeutic. Mol Pharmaceutics 2012, 9: 2669–2676.View ArticleGoogle Scholar
- Dopieralski P, Ribas-Arino J, Anjukandi P, Krupicka M, Kiss J, Marx D: The Janus-faced role of external forces in mechanochemical disulfide bond cleavage. Nat Chem 2013, 5: 685–691.View ArticleGoogle Scholar
- Kuan SL, Ng DYW, Wu Y, Förtsch C, Barth H, Doroshenko M, Koynov K, Meier C, Weil T: pH responsive Janus-like supramolecular fusion proteins for functional protein delivery. J Am Chem Soc 2013, 135: 17254–17257.View ArticleGoogle Scholar
- Wilson SB, Delovitch TL: Janus-like role of regulatory iNKT cells in autoimmune disease and tumour immunity. Nat Rev Immunol 2003, 3: 211–222.View ArticleGoogle Scholar
- Colussi TM, Costantino DA, Hammond JA, Ruehle GM, Nix JC, Kieft JS: The structural basis of transfer RNA mimicry and conformational plasticity by a viral RNA. Nature 2014, 511: 366–369.View ArticleGoogle Scholar
- Chan XWA, Wrenger C, Stahl K, Bergmann B, Winterberg M, Müller IB, Saliba KJ: Chemical and genetic validation of thiamine utilization as an antimalarial drug target. Nat Commun 2013, 4: 2060.View ArticleGoogle Scholar
- Smith CC, Wang Q, Chin CS, Salerno S, Damon LE, Levis MJ, Perl AE, Travers KJ, Wang S, Hunt JP, Zarrinkar PP, Schadt EE, Kasarskis A, Kuriyan J, Shah NP: Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature 2012, 485: 260–263.View ArticleGoogle Scholar
- Salvador-Morales C, Zhang L, Langer R, Farokhzad OC: Immunocompatibility properties of lipid-polymer hybrid nanoparticles with heterogeneous surface functional groups. Biomaterials 2009, 30: 2231–2240.View ArticleGoogle Scholar
- Kievit FM, Zhang M: Cancer nanotheranostics: improving imaging and therapy by targeted delivery across biological barriers. Adv Mater 2011, 23: H217-H247.View ArticleGoogle Scholar
- Alexis F, Pridgen E, Molnar LK, Farokhzad OC: Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol Pharmaceutics 2008, 5: 505–515.View ArticleGoogle Scholar
- Petros RA, DeSimone JM: Strategies in the design of nanoparticles for therapeutic applications. Nat Rev Drug Discovery 2010, 9: 615–627.View ArticleGoogle Scholar
- Leroueil PR, Berry SA, Duthie K, Han G, Rotello VM, McNerny DQ, Baker JR Jr, Orr BG, Holl MM: Wide varieties of cationic nanoparticles induce defects in supported lipid bilayers. Nano Lett 2008, 8: 420–424.View ArticleGoogle Scholar
- Poole AR, Tiltman KJ, Recklies AD, Stoker TAM: Differences in secretion of the proteinase cathepsin B at the edges of human breast carcinomas and fibroadenomas. Nature 1978, 273: 545–547.View ArticleGoogle Scholar
- Moghimi SM, Hunter AC, Murray JC: Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol Rev 2001, 53: 283–318.Google Scholar
- Sibrian-Vazquez M, Jensen TJ, Vicente MG: Synthesis, characterization, and metabolic stability of porphyrin-peptide conjugates bearing bifunctional signaling sequences. J Med Chem 2008, 51: 2915–2923.View ArticleGoogle Scholar
- Romberg B, Hennink W, Storm G: Sheddable coatings for long-circulating nanoparticles. Pharm Res 2008, 25: 55–71.View ArticleGoogle Scholar
- Kohler N, Sun C, Wang J, Zhang M: Methotrexate-modified superparamagnetic nanoparticles and their intracellular uptake into human cancer cells. Langmuir 2005, 21: 8858–8864.View ArticleGoogle Scholar
- Samori C, Ali-Boucetta H, Sainz R, Guo C, Toma FM, Fabbro C, da Ros T, Prato M, Kostarelos K, Bianco A: Enhanced anticancer activity of multi-walled carbon nanotube-methotrexate conjugates using cleavable linkers. Chem Commun 2010, 46: 1494–1496.View ArticleGoogle Scholar
- Rai P, Padala C, Poon V, Saraph A, Basha S, Kate S, Tao K, Mogridge J, Kane RS: Statistical pattern matching facilitates the design of polyvalent inhibitors of anthrax and cholera toxins. Nat Biotechnol 2006, 24: 582–586.View ArticleGoogle Scholar
- Ashley CE, Carnes EC, Phillips GK, Padilla D, Durfee PN, Brown PA, Hanna TN, Liu J, Phillips B, Carter MB, Carroll NJ, Jiang X, Dunphy DR, Willman CL, Petsev DN, Evans DG, Parikh AN, Chackerian B, Wharton W, Peabody DS, Brinker CJ: The targeted delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers. Nat Mater 2011, 10: 389–397.View ArticleGoogle Scholar
- Jiang W, KimBetty YS, Rutka JT, ChanWarren CW: Nanoparticle-mediated cellular response is size-dependent. Nat Nanotechnol 2008, 3: 145–150.View ArticleGoogle Scholar
- Mammen M, Choi S-K, Whitesides GM: Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed 1998, 37: 2754–2794.View ArticleGoogle Scholar
- Pastan I, Hassan R, Fitzgerald DJ, Kreitman RJ: Immunotoxin therapy of cancer. Nat Rev Cancer 2006, 6: 559–565.View ArticleGoogle Scholar
- Licata NA, Tkachenko AV: Kinetic limitations of cooperativity-based drug delivery systems. Phys Rev Lett 2008, 100: 158102–158105.View ArticleGoogle Scholar
- Martinez-Veracoechea FJ, Frenkel D: Designing super selectivity in multivalent nano-particle binding. Proc Natl Acad Sci U S A 2011, 108: 10963–10968.View ArticleGoogle Scholar
- Wang S, Dormidontova EE: Selectivity of ligand-receptor interactions between nanoparticle and cell surfaces. Phys Rev Lett 2012, 109: 238102.View ArticleGoogle Scholar
- Jin E, Zhang B, Sun X, Zhou Z, Ma X, Sun Q, Tang J, Shen Y, Van Kirk E, Murdoch WJ, Radosz M: Acid-active cell-penetrating peptides for in vivo tumor-targeted drug delivery. J Am Chem Soc 2013, 135: 933–940.View ArticleGoogle Scholar
- Mohapatra S, Rout SR, Maiti S, Maiti TK, Panda AB: Monodisperse mesoporous cobalt ferrite nanoparticles: synthesis and application in targeted delivery of antitumor drugs. J Mater Chem 2011, 21: 9185–9193.View ArticleGoogle Scholar
- Richard I, Thibault M, De Crescenzo G, Buschmann MD, Lavertu M: Ionization behavior of chitosan and chitosan-DNA polyplexes indicate that chitosan Has a similar capability to induce a proton-sponge effect as PEI. Biomacromolecules 2013, 14: 1732–1740.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.